Epitope sequences
Disclosed herein are polypeptides, including epitopes, clusters, and antigens. Also disclosed are compositions that include said polypeptides and methods for their use.
[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 60/282,211, filed on Apr. 6, 2001; U.S. Provisional Patent Application Serial No. 60/337,017, filed on Nov. 7, 2001; and U.S. Provisional Patent Application Serial No. 60/363,210, filed on Mar. 7, 2002; all entitled “EPITOPE SEQUENCES,” and all of which are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION[0002] 1. Field of the Invention
[0003] The present invention generally relates to peptides, and nucleic acids encoding peptides, that are useful epitopes of target-associated antigens. More specifically, the invention relates to epitopes that have a high affinity for MHC class I and that are produced by target-specific proteasomes.
[0004] 2. Description of the Related Art
[0005] Neoplasia and the Immune System
[0006] The neoplastic disease state commonly known as cancer is thought to result generally from a single cell growing out of control. The uncontrolled growth state typically results from a multi-step process in which a series of cellular systems fail, resulting in the genesis of a neoplastic cell. The resulting neoplastic cell rapidly reproduces itself, forms one or more tumors, and eventually may cause the death of the host.
[0007] Because the progenitor of the neoplastic cell shares the host's genetic material, neoplastic cells are largely unassailed by the host's immune system. During immune surveillance, the process in which the host's immune system surveys and localizes foreign materials, a neoplastic cell will appear to the host's immune surveillance machinery as a “self” cell.
[0008] Viruses and the Immune System
[0009] In contrast to cancer cells, virus infection involves the expression of clearly non-self antigens. As a result, many virus infections are successfully dealt with by the immune system with minimal clinical sequela. Moreover, it has been possible to develop effective vaccines for many of those infections that do cause serious disease. A variety of vaccine approaches have been used successfully to combat various diseases. These approaches include subunit vaccines consisting of individual proteins produced through recombinant DNA technology. Notwithstanding these advances, the selection and effective administration of minimal epitopes for use as viral vaccines has remained problematic.
[0010] In addition to the difficulties involved in epitope selection stands the problem of viruses that have evolved the capability of evading a host's immune system. Many viruses, especially viruses that establish persistent infections, such as members of the herpes and pox virus families, produce immunomodulatory molecules that permit the virus to evade the host's immune system. The effects of these immunomodulatory molecules on antigen presentation may be overcome by the targeting of select epitopes for administration as immunogenic compositions. To better understand the interaction of neoplastic cells and virally infected cells with the host's immune system, a discussion of the system's components follows below.
[0011] The immune system functions to discriminate molecules endogenous to an organism (“self” molecules) from material exogenous or foreign to the organism (“non-self” molecules). The immune system has two types of adaptive responses to foreign bodies based on the components that mediate the response: a humoral response and a cell-mediated response. The humoral response is mediated by antibodies, while the cell-mediated response involves cells classified as lymphocytes. Recent anticancer and antiviral strategies have focused on mobilizing the host immune system as a means of anticancer or antiviral treatment or therapy.
[0012] The immune system functions in three phases to protect the host from foreign bodies: the cognitive phase, the activation phase, and the effector phase. In the cognitive phase, the immune system recognizes and signals the presence of a foreign antigen or invader in the body. The foreign antigen can be, for example, a cell surface marker from a neoplastic cell or a viral protein. Once the system is aware of an invading body, antigen specific cells of the immune system proliferate and differentiate in response to the invader-triggered signals. The last stage is the effector stage in which the effector cells of the immune system respond to and neutralize the detected invader.
[0013] An array of effector cells implements an immune response to an invader. One type of effector cell, the B cell, generates antibodies targeted against foreign antigens encountered by the host. In combination with the complement system, antibodies direct the destruction of cells or organisms bearing the targeted antigen. Another type of effector cell is the natural killer cell (NK cell), a type of lymphocyte having the capacity to spontaneously recognize and destroy a variety of virus infected cells as well as malignant cell types. The method used by NK cells to recognize target cells is poorly understood.
[0014] Another type of effector cell, the T cell, has members classified into three subcategories, each playing a different role in the immune response. Helper T cells secrete cytokines which stimulate the proliferation of other cells necessary for mounting an effective immune response, while suppressor T cells down-regulate the immune response. A third category of T cell, the cytotoxic T cell (CTL), is capable of directly lysing a targeted cell presenting a foreign antigen on its surface.
[0015] The Major Histocompatibility Complex and T Cell Target Recognition
[0016] T cells are antigen-specific immune cells that function in response to specific antigen signals. B lymphocytes and the antibodies they produce are also antigen-specific entities. However, unlike B lymphocytes, T cells do not respond to antigens in a free or soluble form. For a T cell to respond to an antigen, it requires the antigen to be processed to peptides which are then bound to a presenting structure encoded in the major histocompatibility complex (MHC). This requirement is called “MHC restriction” and it is the mechanism by which T cells differentiate “self” from “non-self” cells. If an antigen is not displayed by a recognizable MHC molecule, the T cell will not recognize and act on the antigen signal. T cells specific for a peptide bound to a recognizable MHC molecule bind to these MHC-peptide complexes and proceed to the next stages of the immune response.
[0017] There are two types of MHC, class I MHC and class II MHC. T Helper cells (CD4+) predominately interact with class II MHC proteins while cytolytic T cells (CD8+) predominately interact with class I MHC proteins. Both classes of MHC protein are transmembrane proteins with a majority of their structure on the external surface of the cell. Additionally, both classes of MHC proteins have a peptide binding cleft on their external portions. It is in this cleft that small fragments of proteins, endogenous or foreign, are bound and presented to the extracellular environment.
[0018] Cells called “professional antigen presenting cells” (pAPCs) display antigens to T cells using the MHC proteins but additionally express various co-stimulatory molecules depending on the particular state of differentiation/activation of the pAPC. When T cells, specific for the peptide bound to a recognizable MHC protein, bind to these MHC-peptide complexes on pAPCs, the specific co-stimulatory molecules that act upon the T cell direct the path of differentiation/activation taken by the T cell. That is, the co-stimulation molecules affect how the T cell will act on antigenic signals in future encounters as it proceeds to the next stages of the immune response.
[0019] As discussed above, neoplastic cells are largely ignored by the immune system. A great deal of effort is now being expended in an attempt to harness a host's immune system to aid in combating the presence of neoplastic cells in a host. One such area of research involves the formulation of anticancer vaccines.
[0020] Anticancer Vaccines
[0021] Among the various weapons available to an oncologist in the battle against cancer is the immune system of the patient. Work has been done in various attempts to cause the immune system to combat cancer or neoplastic diseases. Unfortunately, the results to date have been largely disappointing. One area of particular interest involves the generation and use of anticancer vaccines.
[0022] To generate a vaccine or other immunogenic composition, it is necessary to introduce to a subject an antigen or epitope against which an immune response may be mounted. Although neoplastic cells are derived from and therefore are substantially identical to normal cells on a genetic level, many neoplastic cells are known to present tumor-associated antigens (TuAAs). In theory, these antigens could be used by a subject's immune system to recognize these antigens and attack the neoplastic cells. In reality, however, neoplastic cells generally appear to be ignored by the host's immune system.
[0023] A number of different strategies have been developed in an attempt to generate vaccines with activity against neoplastic cells. These strategies include the use of tumor-associated antigens as immunogens. For example, U.S. Pat. No. 5,993,828, describes a method for producing an immune response against a particular subunit of the Urinary Tumor Associated Antigen by administering to a subject an effective dose of a composition comprising inactivated tumor cells having the Urinary Tumor Associated Antigen on the cell surface and at least one tumor associated antigen selected from the group consisting of GM-2, GD-2, Fetal Antigen and Melanoma Associated Antigen. Accordingly, this patent describes using whole, inactivated tumor cells as the immunogen in an anticancer vaccine.
[0024] Another strategy used with anticancer vaccines involves administering a composition containing isolated tumor antigens. In one approach, MAGE-A1 antigenic peptides were used as an immunogen. (See Chaux, P., et al., “Identification of Five MAGE-A1 Epitopes Recognized by Cytolytic T Lymphocytes Obtained by In Vitro Stimulation with Dendritic Cells Transduced with MAGE-A1,” J. Immunol., 163(5):2928-2936 (1999)). There have been several therapeutic trials using MAGE-A1 peptides for vaccination, although the effectiveness of the vaccination regimes was limited. The results of some of these trials are discussed in Vose, J. M., “Tumor Antigens Recognized by T Lymphocytes,” 10th European Cancer Conference, Day 2, Sept. 14, 1999.
[0025] In another example of tumor associated antigens used as vaccines, Scheinberg, et al. treated 12 chronic myelogenous leukemia (CML) patients already receiving interferon (IFN) or hydroxyurea with 5 injections of class I-associated bcr-abl peptides with a helper peptide plus the adjuvant QS-21. Scheinberg, D. A., et al, “BCR-ABL Breakpoint Derived Oncogene Fusion Peptide Vaccines Generate Specific Immune Responses in Patients with Chronic Myelogenous Leukemia (CML) [Abstract 1665], American Society of Clinical Oncology 35h Annual Meeting, Atlanta (1999). Proliferative and delayed type hypersensitivity (DTH) T cell responses indicative of T-helper activity were elicited, but no cytolytic killer T cell activity was observed within the fresh blood samples.
[0026] Additional examples of attempts to identify TuAAs for use as vaccines are seen in the recent work of Cebon, et al. and Scheibenbogen, et al. Cebon, et al. immunized patients with metastatic melanoma using intradermallly administered MART-126-35 peptide with IL-12 in increasing doses given either subcutaneously or intravenously. Of the first 15 patients, 1 complete remission, 1 partial remission, and 1 mixed response were noted. Immune assays for T cell generation included DTH, which was seen in patients with or without IL-12. Positive CTL assays were seen in patients with evidence of clinical benefit, but not in patients without tumor regression. Cebon, et al., “Phase I Studies of Immunization with Melan-A and IL-12 in HLA A2+ Positive Patients with Stage III and IV Malignant Melanoma,” [Abstract 1671], American Society of Clinical Oncology 35th Annual Meeting, Atlanta (1999).
[0027] Scheibenbogen, et al. immunized 18 patients with 4 HLA class I restricted tyrosinase peptides, 16 with metastatic melanoma and 2 adjuvant patients. Scheibenbogen, et al., “Vaccination with Tyrosinase peptides and GM-CSF in Metastatic Melanoma: a Phase II Trial,” [Abstract 1680], American Society of Clinical Oncology 35h Annual Meeting, Atlanta (1999). Increased CTL activity was observed in 4/15 patients, 2 adjuvant patients, and 2 patients with evidence of tumor regression. As in the trial by Cebon, et al., patients with progressive disease did not show boosted immunity. In spite of the various efforts expended to date to generate efficacious anticancer vaccines, no such composition has yet been developed.
[0028] Antiviral Vaccines
[0029] Vaccine strategies to protect against viral diseases have had many successes. Perhaps the most notable of these is the progress that has been made against the disease small pox, which has been driven to extinction. The success of the polio vaccine is of a similar magnitude.
[0030] Viral vaccines can be grouped into three classifications: live attenuated virus vaccines, such as vaccinia for small pox, the Sabin poliovirus vaccine, and measles mumps and rubella; whole killed or inactivated virus vaccines, such as the Salk poliovirus vaccine, hepatitis A virus vaccine and the typical influenza virus vaccines; and subunit vaccines, such as hepatitis B. Due to their lack of a complete viral genome, subunit vaccines offer a greater degree of safety than those based on whole viruses.
[0031] The paradigm of a successful subunit vaccine is the recombinant hepatitis B vaccine based on the viruses envelope protein. Despite much academic interest in pushing the reductionist subunit concept beyond single proteins to individual epitopes, the efforts have yet to bear much fruit. Viral vaccine research has also concentrated on the induction of an antibody response although cellular responses also occur. However, many of the subunit formulations are particularly poor at generating a CTL response.
SUMMARY OF THE INVENTION[0032] Previous methods of priming professional antigen presenting cells (pAPCs) to display target cell epitopes have relied simply on causing the pAPCs to express target-associated antigens (TAAs), or epitopes of those antigens which are thought to have a high affinity for MHC I molecules. However, the proteasomal processing of such antigens results in presentation of epitopes on the pAPC that do not correspond to the epitopes present on the target cells.
[0033] Using the knowledge that an effective cellular immune response requires that pAPCs present the same epitope that is presented by the target cells, the present invention provides epitopes that have a high affinity for MHC I, and that correspond to the processing specificity of the housekeeping proteasome, which is active in peripheral cells. These epitopes thus correspond to those presented on target cells. The use of such epitopes in vaccines can activate the cellular immune response to recognize the correctly processed TAA and can result in removal of target cells that present such epitopes. In some embodiments, the housekeeping epitopes provided herein can be used in combination with immune epitopes, generating a cellular immune response that is competent to attack target cells both before and after interferon induction. In other embodiments the epitopes are useful in the diagnosis and monitoring of the target-associated disease and in the generation of immunological reagents for such purposes.
[0034] Embodiments of the invention relate to isolated epitopes, and antigens or polypeptides that comprise the epitopes. Preferred embodiments include an epitope or antigen having the sequence as disclosed in Table 1. Other embodiments can include an epitope cluster comprising a polypeptide from Table 1. Further, embodiments include a polypeptide having substantial similarity to the already mentioned epitopes, polypeptides, antigens, or clusters. Other preferred embodiments include a polypeptide having functional similarity to any of the above. Still further embodiments relate to a nucleic acid encoding the polypeptide of any of the epitopes, clusters, antigens, and polypeptides from Table 1 and mentioned herein. For purposes of the following summary, discussions of other embodiments of the invention, when making reference to “the epitope,” or “the epitopes” may refer without limitation to all of the foregoing forms of the epitope.
[0035] The epitope can be immunologically active. The polypeptide comprising the epitope can be less than about 30 amino acids in length, more preferably, the polypeptide is 8 to 10 amino acids in length, for example. Substantial or functional similarity can include addition of at least one amino acid, for example, and the at least one additional amino acid can be at an N-terminus of the polypeptide. The substantial or functional similarity can include a substitution of at least one amino acid.
[0036] The epitope, cluster, or polypeptide comprising the same can have affinity to an HLA-A2 molecule. The affinity can be determined by an assay of binding, by an assay of restriction of epitope recognition, by a prediction algorithm, and the like. The epitope, cluster, or polypeptide comprising the same can have affinity to an HLA-B7, HLA-B51 molecule, and the like.
[0037] In preferred embodiments the polypeptide can be a housekeeping epitope. The epitope or polypeptide can correspond to an epitope displayed on a tumor cell, to an epitope displayed on a neovasculature cell, and the like. The epitope or polypeptide can be an immune epitope. The epitope, cluster and/or polypeptide can be a nucleic acid.
[0038] Other embodiments relate to pharmaceutical compositions comprising the polypeptides, including an epitope from Table 1, a cluster, or a polypeptide comprising the same, and a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, and the like. The adjuvant can be a polynucleotide. The polynucleotide can include a dinucleotide, which can be CpG, for example. The adjuvant can be encoded by a polynucleotide. The adjuvant can be a cytokine and the cytokine can be, for example, GM-CSF.
[0039] The pharmaceutical compositions can further include a professional antigen-presenting cell (pAPC). The pAPC can be a dendritic cell, for example. The pharmaceutical composition can further include a second epitope. The second epitope can be a polypeptide, a nucleic acid, a housekeeping epitope, an immune epitope, and the like.
[0040] Still further embodiments relate to pharmaceutical compositions that include any of the nucleic acids discussed herein, including those that encode polypeptides that comprise epitopes or antigens from Table 1. Such compositions can include a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, and the like.
[0041] Other embodiments relate to recombinant constructs that include such a nucleic acid as described herein, including those that encode polypeptides that comprise epitopes or antigens from Table 1. The constructs can further include a plasmid, a viral vector, an artificial chromosome, and the like. The construct can further include a sequence encoding at least one feature, such as for example, a second epitope, an IRES, an ISS, an NIS, a ubiquitin, and the like.
[0042] Further embodiments relate to purified antibodies that specifically bind to at least one of the epitopes in Table 1. Other embodiments relate to purified antibodies that specifically bind to a peptide-MHC protein complex comprising an epitope disclosed in Table 1 or any other suitable epitope. The antibody from any embodiment can be a monoclonal antibody or a polyclonal antibody.
[0043] Still other embodiments relate to multimeric MHC-peptide complexes that include an epitope, such as, for example, an epitope disclosed in Table 1. Also, contemplated are antibodies specific for the complexes.
[0044] Embodiments relate to isolated T cells expressing a T cell receptor specific for an MHC-peptide complex. The complex can include an epitope, such as, for example, an epitope disclosed in Table 1. The T cell can be produced by an in vitro immunization and can be isolated from an immunized animal. Embodiments relate to T cell clones, including cloned T cells, such as those discussed above. Embodiments also relate to polyclonal population of T cells. Such populations can include a T cell, as described above, for example.
[0045] Still further embodiments relate to pharmaceutical compositions that include a T cell, such as those described above, for example, and a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, and the like.
[0046] Embodiments of the invention relate to isolated protein molecules comprising the binding domain of a T cell receptor specific for an MHC-peptide complex. The complex can include an epitope as disclosed in Table 1. The protein can be multivalent. Other embodiments relate to isolated nucleic acids encoding such proteins. Still further embodiments relate to recombinant constructs that include such nucleic acids.
[0047] Other embodiments of the invention relate to host cells expressing a recombinant construct as described herein, including constructs encoding an epitope, cluster or polypeptide comprising the same, disclosed in Table 1, for example. The host cell can be a dendritic cell, macrophage, tumor cell, tumor-derived cell, a bacterium, fungus, protozoan, and the like. Embodiments also relate to pharmaceutical compositions that include a host cell, such as those discussed herein, and a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, and the like.
[0048] Still other embodiments relate to vaccines or immunotherapeutic compositions that include at least one component, such as, for example, an epitope disclosed in Table 1 or otherwise described herein; a cluster that includes such an epitope, an antigen or polypeptide that includes such an epitope; a composition as described above and herein; a construct as described above and herein, a T cell, or a host cell as described above and herein.
[0049] Further embodiments relate to methods of treating an animal. The methods can include administering to an animal a pharmaceutical composition, such as, a vaccine or immunotherapeutic composition, including those disclosed above and herein. The administering step can include a mode of delivery, such as, for example, transdermal, intranodal, perinodal, oral, intravenous, intradermal, intramuscular, intraperitoneal, mucosal, aerosol inhalation, instillation, and the like. The method can further include a step of assaying to determine a characteristic indicative of a state of a target cell or target cells. The method can include a first assaying step and a second assaying step, wherein the first assaying step precedes the administering step, and wherein the second assaying step follows the administering step. The method can further include a step of comparing the characteristic determined in the first assaying step with the characteristic determined in the second assaying step to obtain a result. The result can be for example, evidence of an immune response, a diminution in number of target cells, a loss of mass or size of a tumor comprising target cells, a decrease in number or concentration of an intracellular parasite infecting target cells, and the like.
[0050] Embodiments relate to methods of evaluating immunogenicity of a vaccine or immunotherapeutic composition. The methods can include administering to an animal a vaccine or immunotherapeutic, such as those described above and elsewhere herein, and evaluating immunogenicity based on a characteristic of the animal. The animal can be HLA-transgenic.
[0051] Other embodiments relate to methods of evaluating immunogenicity that include in vitro stimulation of a T cell with the vaccine or immunotherapeutic composition, such as those described above and elsewhere herein, and evaluating immunogenicity based on a characteristic of the T cell. The stimulation can be a primary stimulation.
[0052] Still further embodiments relate to methods of making a passive/adoptive immunotherapeutic. The methods can include combining a T cell or a host cell, such as those described above and elsewhere herein, with a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, and the like.
[0053] Other embodiments relate to methods of determining specific T cell frequency, and can include the step of contacting T cells with a MHC-peptide complex comprising an epitope disclosed in Table 1, or a complex comprising a cluster or antigen comprising such an epitope. The contacting step can include at least one feature, such as, for example, immunization, restimulation, detection, enumeration, and the like. The method can further include ELISPOT analysis, limiting dilution analysis, flow cytometry, in situ hybridization, the polymerase chain reaction, any combination thereof, and the like.
[0054] Embodiments relate to methods of evaluating immunologic response. The methods can include the above-described methods of determining specific T cell frequency carried out prior to and subsequent to an immunization step.
[0055] Other embodiments relate to methods of evaluating immunologic response. The methods can include determining frequency, cytokine production, or cytolytic activity of T cells, prior to and subsequent to a step of stimulation with MHC-peptide complexes comprising an epitope, such as, for example an epitope from Table 1, a cluster or a polypeptide comprising such an epitope.
[0056] Further embodiments relate to methods of diagnosing a disease. The methods can include contacting a subject tissue with at least one component, including, for example, a T cell, a host cell, an antibody, a protein, including those described above and elsewhere herein; and diagnosing the disease based on a characteristic of the tissue or of the component. The contacting step can take place in vivo or in vitro, for example.
[0057] Still other embodiments relate to methods of making a vaccine. The methods can include combining at least one component, an epitope, a composition, a construct, a T cell, a host cell; including any of those described above and elsewhere herein, with a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, and the like.
[0058] Embodiments relate to computer readable media having recorded thereon the sequence of any one of SEQ ID NOS: 1-602, in a machine having a hardware or software that calculates the physical, biochemical, immunologic, molecular genetic properties of a molecule embodying said sequence, and the like.
[0059] Still other embodiments relate to methods of treating an animal. The methods can include combining the method of treating an animal that includes administering to the animal a vaccine or immunotherapeutic composition, such as described above and elsewhere herein, combined with at least one mode of treatment, including, for example, radiation therapy, chemotherapy, biochemotherapy, surgery, and the like.
[0060] Further embodiments relate to isolated polypeptides that include an epitope cluster. In preferred embodiments the cluster can be from a target-associated antigen having the sequence as disclosed in any one of Tables 25-44, wherein the amino acid sequence includes not more than about 80% of the amino acid sequence of the antigen. Other embodiments relate to vaccines or immunotherapeutic products that include an isolated peptide as described above and elsewhere herein. Still other embodiments relate to isolated polynucleotides encoding a polypeptide as described above and elsewhere herein. Other embodiments relate vaccines or immunotherapeutic products that include these polynucleotides. The polynucleotide can be DNA, RNA, and the like.
[0061] Still further embodiments relate to kits comprising a delivery device and any of the embodiments mentioned above and elsewhere herein. The delivery device can be a catheter, a syringe, an internal or external pump, a reservoir, an inhaler, microinjector, a patch, and any other like device suitable for any route of delivery. As mentioned, the kit, in addition to the delivery device also includes any of the embodiments disclosed herein. For example, without limitations, the kit can include an isolated epitope, a polypeptide, a cluster, a nucleic acid, an antigen, a pharmaceutical composition that includes any of the foregoing, an antibody, a T cell, a T cell receptor, an epitope-MHC complex, a vaccine, an immunotherapeutic, and the like. The kit can also include items such as detailed instructions for use and any other like item.
BRIEF DESCRIPTION OF THE DRAWINGS[0062] FIG. 1 is a sequence alignment of NY-ESO-1 and several similar protein sequences.
[0063] FIG. 2 graphically represents a plasmid vaccine backbone useful for delivering nucleic acid-encoded epitopes.
[0064] FIGS. 3A and 3B are FACS profiles showing results of HLA-A2 binding assays for tyrosinase207-215 and tyrosinase208-216.
[0065] FIG. 3C shows cytolytic activity against a tyrosinase epitope by human CTL induced by in vitro immunization.
[0066] FIG. 4 is a T=120 min. time point mass spectrum of the fragments produced by proteasomal cleavage of SSX-231-68.
[0067] FIG. 5 shows a binding curve for HLA-A2:SSX-241-49 with controls.
[0068] FIG. 6 shows specific lysis of SSX-241-49-pulsed targets by CTL from SSX-241-49-immunized HLA-A2 transgenic mice.
[0069] FIG. 7A, B, and C show results of N-terminal pool sequencing of a T=60 min. time point aliquot of the PSMA163-192 proteasomal digest.
[0070] FIG. 8 shows binding curves for HLA-A2:PSMA168-177 and HLA-A2:PSMA288-297 with controls.
[0071] FIG. 9 shows results of N-terminal pool sequencing of a T=60 min. time point aliquot of the PSMA281-310 proteasomal digest.
[0072] FIG. 10 shows binding curves for HLA-A2:PSMA461-469, HLA-A2:PSMA460-469, and HLA-A2:PSMA663-671, with controls.
[0073] FIG. 11 shows the results of a &ggr;-IFN-based ELISPOT assay detecting PSMA463-471-reactive HLA-A1+ CD8+ T cells.
[0074] FIG. 12 shows blocking of reactivity of the T cells used in FIG. 10 by anti-HLA-A1 mAb, demonstrating HLA-A1-restricted recognition.
[0075] FIG. 13 shows a binding curve for HLA-A2:PSMA663-671, with controls.
[0076] FIG. 14 shows a binding curve for HLA-A2:PSMA662-671, with controls.
[0077] FIG. 15. Comparison of anti-peptide CTL responses following immunization with various doses of DNA by different routes of injection.
[0078] FIG. 16. Growth of transplanted gp33 expressing tumor in mice immunized by i.ln. injection of gp33 epitope-expressing, or control, plasmid.
[0079] FIG. 17. Amount of plasmid DNA detected by real-time PCR in injected or draining lymph nodes at various times after i.ln. of i.m. injection, respectively.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT[0080] Definitions
[0081] Unless otherwise clear from the context of the use of a term herein, the following listed terms shall generally have the indicated meanings for purposes of this description.
[0082] PROFESSIONAL ANTIGEN-PRESENTING CELL (pAPC)—a cell that possesses T cell costimulatory molecules and is able to induce a T cell response. Well characterized pAPCs include dendritic cells, B cells, and macrophages.
[0083] PERIPHERAL CELL—a cell that is not a pAPC.
[0084] HOUSEKEEPING PROTEASOME—a proteasome normally active in peripheral cells, and generally not present or not strongly active in pAPCs.
[0085] IMMUNE PROTEASOME—a proteasome normally active in pAPCs; the immune proteasome is also active in some peripheral cells in infected tissues.
[0086] EPITOPE—a molecule or substance capable of stimulating an immune response. In preferred embodiments, epitopes according to this definition include but are not necessarily limited to a polypeptide and a nucleic acid encoding a polypeptide, wherein the polypeptide is capable of stimulating an immune response. In other preferred embodiments, epitopes according to this definition include but are not necessarily limited to peptides presented on the surface of cells, the peptides being non-covalently bound to the binding cleft of class I MHC, such that they can interact with T cell receptors.
[0087] MHC EPITOPE—a polypeptide having a known or predicted binding affinity for a mammalian class I or class II major histocompatibility complex (MHC) molecule.
[0088] HOUSEKEEPING EPITOPE—In a preferred embodiment, a housekeeping epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which housekeeping proteasomes are predominantly active. In another preferred embodiment, a housekeeping epitope is defined as a polypeptide containing a housekeeping epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, a housekeeping epitope is defined as a nucleic acid that encodes a housekeeping epitope according to the foregoing definitions.
[0089] IMMUNE EPITOPE—In a preferred embodiment, an immune epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which immune proteasomes are predominantly active. In another preferred embodiment, an immune epitope is defined as a polypeptide containing an immune epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, an immune epitope is defined as a polypeptide including an epitope cluster sequence, having at least two polypeptide sequences having a known or predicted affinity for a class I MHC. In yet another preferred embodiment, an immune epitope is defined as a nucleic acid that encodes an immune epitope according to any of the foregoing definitions.
[0090] TARGET CELL—a cell to be targeted by the vaccines and methods of the invention. Examples of target cells according to this definition include but are not necessarily limited to: a neoplastic cell and a cell harboring an intracellular parasite, such as, for example, a virus, a bacterium, or a protozoan.
[0091] TARGET-ASSOCIATED ANTIGEN (TAA)—a protein or polypeptide present in a target cell.
[0092] TUMOR-ASSOCIATED ANTIGENS (TuAA)—a TAA, wherein the target cell is a neoplastic cell.
[0093] HLA EPITOPE—a polypeptide having a known or predicted binding affinity for a human class I or class II HLA complex molecule.
[0094] ANTIBODY—a natural immunoglobulin (Ig), poly- or monoclonal, or any molecule composed in whole or in part of an Ig binding domain, whether derived biochemically or by use of recombinant DNA. Examples include inter alia, F(ab), single chain Fv, and Ig variable region-phage coat protein fusions.
[0095] ENCODE—an open-ended term such that a nucleic acid encoding a particular amino acid sequence can consist of codons specifying that (poly)peptide, but can also comprise additional sequences either translatable, or for the control of transcription, translation, or replication, or to facilitate manipulation of some host nucleic acid construct.
[0096] SUBSTANTIAL SIMILARITY—this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of the sequence. Nucleic acid sequences encoding the same amino acid sequence are substantially similar despite differences in degenerate positions or modest differences in length or composition of any non-coding regions. Amino acid sequences differing only by conservative substitution or minor length variations are substantially similar. Additionally, amino acid sequences comprising housekeeping epitopes that differ in the number of N-terminal flanking residues, or immune epitopes and epitope clusters that differ in the number of flanking residues at either terminus, are substantially similar. Nucleic acids that encode substantially similar amino acid sequences are themselves also substantially similar.
[0097] FUNCTIONAL SIMILARITY—this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of a biological or biochemical property, although the sequences may not be substantially similar. For example, two nucleic acids can be useful as hybridization probes for the same sequence but encode differing amino acid sequences. Two peptides that induce cross-reactive CTL responses are functionally similar even if they differ by non-conservative amino acid substitutions (and thus do not meet the substantial similarity definition). Pairs of antibodies, or TCRs, that recognize the same epitope can be functionally similar to each other despite whatever structural differences exist. In testing for functional similarity of immunogenicity one would generally immunize with the “altered” antigen and test the ability of the elicited response (Ab, CTL, cytokine production, etc.) to recognize the target antigen. Accordingly, two sequences may be designed to differ in certain respects while retaining the same function. Such designed sequence variants are among the embodiments of the present invention. 1 TABLE 1A SEQ ID NOS.* including epitopes in Examples 1-7, 13. SEQ ID NO ENTITY SEQUENCE 1 Tyr 207-216 FLPWHRLFLL 2 Tyrosinase Accession number**: P14679 protein 3 SSX-2 protein Accession number: NP_003138 4 PSMA protein Accession number: NP_004467 5 Tyrosinase cDNA Accession number: NM_000372 6 SSX-2 cDNA Accession number: NM_003147 7 PSMA cDNA Accession number: NM_004476 8 Tyr 207-215 LPWHRLFL 9 Tyr 208-216 LPWHRLFLL 10 SSX-2 31-68 YFSKEEWEKMKASEKIFYVYMKRKYEAMT KLGFKATLP 11 SSX-2 32-40 FSKEEWEKM 12 SSX-2 39-47 KMKASEKIF 13 SSX-2 40-48 MKASEKIFY 14 SSX-2 39-48 KMKASEKIFY 15 SSX-2 41-49 KASEKIFYV 16 SSX-2 40-49 MKASEKLFYV 17 SSX-2 41-50 KASEKIFYVY 18 SSX-2 42-49 ASEKIFYVY 19 SSX-2 53-61 RYEAMTKL 20 SSX-2 52-61 KRYEAMTKL 21 SSX-2 54-63 KYEAMTKLGF 22 SSX-2 55-63 YEAMTKLGF 23 SSX-2 56-63 EAMTKLGF 24 HBV18-27 FLPSDYFPSV 25 HLA-B44 binder AEMGKYSFY 26 SSX-141-49 KYSEKJSYV 27 SSX-341-49 KVSEKJVYV 28 SSX-441-49 KSSEKIVYV 29 SSX-541-49 KASEKIIYV 30 PSMA163-192 AFSPQGMPEGDLVYVNYARTEDFFKLERD M 31 PSMA 168-190 GMPEGDLVYVNYARTEDFFKLER 32 PSMA 169-177 MPEGDLVYV 33 PSMA 168-177 GMPEGDLVYV 34 PSMA 168-176 GMPEGDLVY 35 PSMA 167-176 QGMPEGDLVY 36 PSMA 169-176 MPEGDLVY 37 PSMA 171-179 EGDLVYVNY 38 PSMA 170-179 PEGDLVYVNY 39 PSMA 174-183 LVYVNYARTE 40 PSMA 177-185 VNYARTEDF 41 PSMA 176-185 YVNYARTEDF 42 PSMA 178-186 NYARTEDFF 43 PSMA 179-186 YARTEDFF 44 PSMA 181-189 RTEDFFKLE 45 PSMA 281-310 RGIAEAVGLPSIPVHPIGYYDAQKLLEKM G 46 PSMA 283-307 IAEAVGLPSIPVHPIGYYDAQKLLE 47 PSMA 289-297 LPSIPVHPI 48 PSMA 288-297 GLPSIPVHPI 49 PSMA 297-305 IGYYDAQKL 50 PSMA 296-305 PIGYYDAQKL 51 PSMA 291-299 SIPVHPIGY 52 PSMA 290-299 PSIPVHPIGY 53 PSMA 292-299 IPVHPIGY 54 PSMA 299-307 YYDAQKLLE 55 PSMA 454-481 SSIEGNYTLRVDCTPLMYSLVHLTKEL 56 PSMA 456-464 IEGNYTLRV 57 PSMA 455-464 SIEGNYTLRV 58 PSMA 457-464 EGNYTLRV 59 PSMA 461-469 TLRVDCTPL 60 PSMA 460-469 YTLRVDCTPL 61 PSMA 462-470 LRVDCTPLM 62 PSMA 463-471 RVDCTPLMY 63 PSMA 462-471 LRVDCTPLMY 64 PSMA 653-687 FDKSNPIVLRMMNDQLMFLERAFIDPLGL PDRPFY 65 PSMA 660-681 VLRMMNDQLMFLERAFIIDPLGL 66 PSMA 663-671 MMNDQLMFL 67 PSMA 662-671 RMMNDQLMFL 68 PSMA 662-670 RMMNDQLMF 69 Tyr 1-17 MLLAVLYCLLWSFQTSA
[0098] 2 TABLE 1B SEQ ID NOS.* including epitopes in Examples 14 and 15. SEQ ID NO ENTITY SEQUENCE 70 GP100 protein2 **Accession number: P40967 71 MAGE-1 protein Accession number: P43355 72 MAGE-2 protein Accession number: P43356 73 MAGE-3 protein Accession number: P43357 74 NY-ESO-1 protein Accession number: P78358 75 LAGE-1a protein Accession number: CAA11116 76 LAGE-1b protein Accession number: CAA11117 77 PRAME protein Accession number: NP 006106 78 PSA protein Accession number: P07288 79 PSCA protein Accession number: O43653 80 GP100 cds Accession number: U20093 81 MAGE-1 cds Accession number: M77481 82 MAGE-2 cds Accession number: L18920 83 MAGE-3 cds Accession number: U03735 84 NY-ESO-1 cDNA Accession number: U87459 85 PRAME cDNA Accession number: NM_006115 86 PSA cDNA Accession number: NM_001648 87 PSCA cDNA Accession number: AE043498 88 GP100 630-638 LPHSSSHWL 89 GP100 629-638 QLPHSSSHWL 90 GP100 614-622 LIYRRRLMK 91 GP100 613-622 SLIYRRRLMK 92 GP100 615-622 TYRRRLMK 93 GP100 630-638 LPHSSSHWL 94 GP100 629-638 QLPHSSSHWL 95 MAGE-1 95-102 ESLFRAVI 96 MAGE-1 93-102 ILESLFRAVI 97 MAGE-1 93-101 ILESLFRAV 98 MAGE-1 92-101 CILESLFRAV 99 MAGE-1 92-100 CILESLFRA 100 MAGE-1 263-271 EFLWGPRAL 101 MAGE-1 264-271 FLWGPRAL 102 MAGE-1 264-273 FLWGPRALAE 103 MAGE-1 265-274 LWGPRALAET 104 MAGE-1 268-276 PRALAETSY 105 MAGE-1 267-276 GPRALAETSY 106 MAGE-1 269-277 RALAETSYV 107 MAGE-1 271-279 LAETSYVKV 108 MAGE-1 270-279 ALAETSYVKV 109 MAGE-1 272-280 AETSYVKVL 110 MAGE-1 271-280 LAETSYVKVL 111 MAGE-1 274-282 TSYVKVLEY 112 MAGE-1 273-282 ETSYVKVLEY 113 MAGE-1 278-286 KVLEYVIKV 114 MAGE-1 168-177 SYVLVTCLGL 115 MAGE-1 169-177 YLVTCLGL 116 MAGE-1 170-177 VLVTCLGL 117 MAGE-1 240-248 TQDLVQEKY 118 MAGE-1 239-248 LTQDLVQEKY 119 MAGE-1 232-240 YGEPRKLLT 120 MAGE-1 243-251 LVQEKYLEY 121 MAGE-1 242-251 DLVQEKYLEY 122 MAGE-1 230-238 SAYGEPRKL 123 MAGE-1 278-286 KVLEYVIKV 124 MAGE-1 277-286 VKVLEYVIKV 125 MAGE-1 276-284 YVKVLEYVI 126 MAGE-1 274-282 TSYVKVLEY 127 MAGE-1 273-282 ETSYVKVLEY 128 MAGE-1 283-291 VIKVSARVR 129 MAGE-1 282-291 YVIKVSARVR 130 MAGE-2 115-122 ELVHFLLL 131 MAGE-2 113-122 MVELVHFLLL 132 MAGE-2 109-116 ISRKMVEL 133 MAGE-2 108-116 AISRKMVEL 134 MAGE-2 107-116 AAISRKMVEL 135 MAGE-2 112-120 KMVELVHIFL 136 MAGE-2 109-117 ISRKMVELV 137 MAGE-2 108-117 AISRKMVELV 138 MAGE-2 116-124 LVHFLLLKY 139 MAGE-2 115-124 ELVHFLLLKY 140 MAGE-2 111-119 RKMVELVHF 141 MAGE-2 158-166 LQLVFGIEV 142 MAGE-2 157-166 YLQLVFGIEV 143 MAGE-2 159-167 QLVFGIEVV 144 MAGE-2 158-167 LQLVFGIEVV 145 MAGE-2 164-172 IEVVEVVPI 146 MAGE-2 163-172 GIEVVEVVPI 147 MAGE-2 162-170 FGIEVVEVV 148 MAGE-2 154-162 ASEYLQLVF 149 MAGE-2 153-162 KASEYLQLVF 150 MAGE-2 218-225 EEKIWEEL 151 MAGE-2 216-225 APEEKIWEEL 152 MAGE-2 216-223 APEEKIWE 153 MAGE-2 220-228 KILWEELSML 154 MAGE-2 219-228 EKIWEELSML 155 MAGE-2 271-278 FLWGPRAL 156 MAGE-2 271-279 FLWGPRALI 157 MAGE-2 278-286 LIETSYVKV 158 MAGE-2 277-286 ALIETSYVKV 159 MAGE-2 276-284 RALIETSYV 160 MAGE-2 279-287 IETSYVKVL 161 MAGE-2 278-287 LIETSYVKVL 162 MAGE-3 271-278 FLWGPRAL 163 MAGE-3 270-278 EFLWGPRAL 164 MAGE-3 271-279 FLWGPRALV 165 MAGE-3 276-284 RALVETSYV 166 MAGE-3 272-280 LWGPRALVE 167 MAGE-3 271-280 FLWGPRALVE 168 MAGE-3 272-281 LWGPRALVET 169 NY-ESO-1 82-90 GPESRLLEF 170 NY-ESO-1 83-91 PESRLLEFY 171 NY-ESO-1 82-91 GPESRLLEFY 172 NY-ESO-1 84-92 ESRLLEFYL 173 NY-ESO-1 86-94 RLLEFYLAM 174 NY-ESO-1 88-96 LEFYLAMPF 175 NY-ESO-1 87-96 LLEFYLAMPF 176 NY-ESO-1 93-102 AMPFATPMEA 177 NY-ESO-1 94-102 MPFATPMEA 178 NY-ESO-1 115-123 PLPVPGVLL 179 NY-ESO-1 114-123 PPLPVPGVLL 180 NY-ESO-1 116-123 LPVPGVLL 181 NY-ESO-1 103-112 ELARRSLAQD 182 NY-ESO-1 118-126 VPGVLLKEF 183 NY-ESO-1 117-126 PVPGVLLKEF 184 NY-ESO-1 116-123 LPVPGVLL 185 NY-ESO-1 127-135 TVSGNILTI 186 NY-ESO-1 126-135 FTVSGNILTI 187 NY-ESO-1 120-128 GVLLKEFTV 188 NY-ESO-1 121-130 VLLKEFTVSG 189 NY-ESO-1 122-130 LLKEFTVSG 190 NY-ESO-1 118-126 VPGVLLKEF 191 NY-ESO-1 117-126 PVPGVLLKEF 192 NY-ESO-1 139-147 AADHRQLQL 193 NY-ESO-1 148-156 SISSCLQQL 194 NY-ESO-1 147-156 LSISSCLQQL 195 NY-ESO-1 138-147 TAADHRQLQL 196 NY-ESO-1 161-169 WITQCFLPV 197 NY-ESO-1 157-165 SLLMW1TQC 198 NY-ESO-1 150-158 SSCLQQLSL 199 NY-ESO-1 154-162 QQLSLLMWI 200 NY-ESO-1 151-159 SCLQQLSLL 201 NY-ESO-1 150-159 SSGLQQLSLL 202 NY-ESO-1 163-171 TQCFLPVFL 203 NY-ESO-1 162-171 ITQCFLPVFL 204 PRAME 219-227 PMQDII(MIL 205 PRAME 218-227 MPMQDIKMIL 206 PRAME 428-436 QHLIGLSNL 207 PRAME 427-436 LQHLIGLSNL 208 PRAME 429-436 HLIGLSNL 209 PRAME 431-439 IGLSNLTHV 210 PRAME 43O-439 LIGLSNLTHV 211 PSA 53-61 VLVHPQWVL 212 PSA 52-61 GVLVHPQWVL 213 PSA 52-60 GVLVHPQWV 214 PSA 59-67 WVLTAAHCI 215 PSA 54-63 LVHPQWVLTA 216 PSA 53-62 VLVHPQWVLT 217 PSA 54-62 LVHPQWVLT 218 PSA 66-73 CIRNKSVI 219 PSA 65-73 HCIRNKSVI 220 PSA 56-64 HPQWVLTAA 221 PSA 63-72 AAHCIRNKSV 222 PSCA 116-123 LLWGPGQL 223 PSCA 115-123 LLLWGPGQL 224 PSCA 114-123 GLLLWGPGQL 225 PSCA 99-107 ALQPAAATL 226 PSCA 98-107 ALQPAAAIL 227 Tyr 128-137 APEKDKFFAY 228 Tyr 129-137 PEKDKFFAY 229 Tyr 130-138 EKDKFFAYL 230 Tyr 131-138 KDKFFAYL 231 Tyr 205-213 PAFLPWHRL 232 Tyr 204-213 APAFLPWHRL 233 Tyr 214-223 FLLRWEQEIQ 234 Tyr 212-220 RLFLLRWEQ 235 Tyr 191-200 GSEIWRDIDF 236 Tyr 192-200 SEIWRDIDF 237 Tyr 473-481 RIWSWLLGA 238 Tyr 476-484 SWLLGAAMV 239 Tyr 477-486 WLLGAAMVGA 240 Tyr 478-486 LLGAAMVGA 241 PSMA 4-12 LLHETDSAV 242 PSMA 13-21 ATARRPRWL 243 PSMA 53-61 TPKHNMKAF 244 PSMA 64-73 ELKAENIKKF 245 PSMA 69-77 NIKKFLH1NF 246 PSMA 68-77 ENIKKFLH1NF 247 PSMA 220-228 AGAKGVILY 248 PSMA 468-477 PLMYSLVHNL 249 PSMA 469-477 LMYSLVHNL 250 PSMA 463-471 RVDCTPLMY 251 PSMA 465-473 DCTPLMYSL 252 PSMA 507-515 SGMPRISKL 253 PSMA 506-515 FSGMPRISKL 254 NY-ESO-1 136-163 RLTAADHRQLQLSJSSCLQQLSLLMWIT 255 NY-ESO-1 150-177 SSCLQQLSLLMWITQCFLPVFLAQPPSG 1This H was reported as Y in the SWISSPROT database. 2The amino acid at position 274 may be Pro or Leu depending upon the database. The particular analysis presented herein used the Pro.
[0099] 3 TABLE 1C SEQ ID NOS.* including epitopes in Example 14. SEQ ID NO. IDENTITY SEQUENCE 256 Mage-1 125-132 KAEMLESV 257 Mage-1 124-132 TKAEMLESV 258 Mage-1 123-132 VTKAEMLESV 259 Mage-1 128-136 MLESVIKNY 260 Mage-1 127-136 EMLESVIKNY 261 Mage-1 125-133 KAEMLESVI 262 Mage-1 146-153 KASESLQL 263 Mage-1 145-153 GKASESLQL 264 Mage-1 147-155 ASESLQLVF 265 Mage-1 153-161 LVFGTDVKE 266 Mage-1 114-121 LLKYRARE 267 Mage-1 106-113 VADLVGFL 268 Mage-1 105-113 KVADLVGFL 269 Mage-1 107-115 ADLVGFLLL 270 Mage-1 106-115 VADLVGFLLL 271 Mage-1 114-123 LLKYRAREPV 272 Mage-3 278-286 LVETSYVKV 273 Mage-3 277-286 ALVETSYVKV 274 Mage-3 285-293 KVLHHMVKI 275 Mage-3 283-291 YVKVLIIIIMV 276 Mage-3 275-283 PRALVETSY 277 Mage-3 274-283 GPRALVETSY 278 Mage-3 278-287 LVETSYVKVL 279 ED-B 4′-5 TIIPEVPQL 280 ED-B 5′-5 DTIIPEVPQL 281 ED-B 1-10 EVPQLTDLSF 282 ED-B 23-30 TPLNSSTI 283 ED-B 18-25 IGLRWTPL 284 ED-B 17-25 SIGLRWTPL 285 ED-B 25-33 LNSSTIIGY 286 ED-B 24-33 PLNSSTIIGY 287 ED-B 23-31 TPLNSSTII 288 ED-B 31-38 IGYRITVV 289 ED-B 30-38 IIGYRITVV 290 ED-B 29-38 TIIGYRJTVV 291 ED-B 31-39 IGXTRITVVA 292 ED-B 30-39 IIGYRITVVA 293 CEA 184-191 SLPVSPRL 294 CEA 183-191 QSLPVSPRL 295 CEA 186-193 PVSPRLQL 296 CEA 185-193 LPVSPRLQL 297 CEA 184-193 SLPVSPRLQL 298 CEA 185-192 LPVSPRLQ 299 GEA 192-200 QLSNGNRTL 300 CEA 191-200 LQLSNGNRTL 301 CEA 179-187 WVNNQSLPV 302 GEA 186-194 PVSPRLQLS 303 CEA 362-369 SLPVSPRL 304 CEA 361-369 QSLPVSPRL 305 CEA 364-371 PVSPRLQL 306 CEA 363-371 LPVSPRLQL 307 CEA 362-371 SLPVSPRLQL 308 CEA 363-370 LPVSPRLQ 309 CEA 370-378 QLSNDNRTL 310 CEA 369-378 LQLSNDNRTL 311 CEA 357-365 WVNNQSLPV 312 CEA 360-368 NQSLPVSPR 313 CEA 540-547 SLPVSPRL 314 CEA 539-547 QSLPVSPRL 315 CEA 542-549 PVSPRLQL 316 CEA 541-549 LPVSPRLQL 317 CEA 540-549 SLPVSPRLQL 318 CEA 541-548 LPVSPRLQ 319 CEA 548-556 QLSNGNRTL 320 CEA 547-556 LQLSNGNRTL 321 CEA 535-543 WVNGQSLPV 322 CEA 533-541 LWWVNGQSL 323 CEA 532-541 YLWWVNGQSL 324 CEA 538-546 GQSLPVSPR 325 Her-2 30-37 DMKLRLPA 326 Her-2 28-37 GTDMKLRLPA 327 Her-2 42-49 HLDMLRITL 328 Her-2 41-49 THLDMLRJIL 329 Her-2 40-49 ETHLDMLRHL 330 Her-2 36-43 PASPETHL 331 Her-2 35-43 LPASPETHL 332 Her-2 34-43 RLPASPETHL 333 Her-2 38-46 SPETHLDML 334 Her-2 37-46 ASPETHLDML 335 Her-2 42-50 HLDMLRIILY 336 Her-2 41-50 THLDMLRIILY 337 Her-2 719-726 ELRKVKVL 338 Her-2 718-726 TELRKVKVL 339 Her-2 717-726 ETELRKVKVL 340 Her-2 715-723 LKETELRKV 341 Her-2 714-723 ILKETELRKiV 342 Her-2 712-720 MR1LKETEL 343 Her-2 711-720 QMRILKETEL 344 Her-2 717-725 ETELRKVKV 345 Her-2 716-725 KETELRKVKV 346 Her-2 706-714 MPNQAQMRI 347 Her-2 705-714 AMPNQAQMRI 348 Her-2 706-715 MPNQAQMRIL 349 HER-2 966-973 RPRFRELV 350 HER-2 965-973 CRPRFRELV 351 HER-2 968-976 RFRELVSEF 352 HER-2 967-976 PRFRELVSEF 353 HER-2 964-972 ECRPRFREL 354 NY-ESO-1 67-75 GAASGLNGC 355 NY-ESO-1 52-60 RASGPGGGA 356 NY-ESO-1 64-72 PHGGAASGL 357 NY-ESO-1 63-72 GPHGGAASGL 358 NY-ESO-1 60-69 APRGPHGGAA 359 PRAME 112-119 VRPRRWKL 360 PRAME 111-119 EVRPRRWKL 361 PRAME 113-121 RPRRWKLQV 362 PRAME 114-122 PRRWKLQVL 363 PRAME 113-122 RPRRWKLQVL 364 PRAME 116-124 RWKLQVLDL 365 PRAME 115-124 RRWKLQVLDL 366 PRAME 174-182 PVEVLVDLF 367 PRAME 199-206 VKRKKNVL 368 PRAME 198-206 KVKRKKNVL 369 PRAME 197-206 EKVKRiKKNVL 370 PRAME 198-205 KVKRKKNV 371 PRAME 201-208 RKKNVLRL 372 PRAME 200-208 KRKKNVLRL 373 PRAME 199-208 VKRKKNVLRL 374 PRAME 189-196 DELESYLI 375 PRAME 205-213 VLRLCCKKL 376 PRAME 204-213 NVLRLGCKKL 377 PRAME 194-202 YLLEKVKRK 378 PRAME 74-81 QAWPFTCL 379 PRAME 73-81 VQAWPFTCL 380 PRAME 72-81 MVQAWPFTCL 381 PRAME 81-88 LPLGVLMK 382 PRAME 80-88 CLPLGVLMK 383 PRAME 79-88 TCLPLGVLMK 384 PRAME 84-92 GVLMKGQHL 385 PRAME 81-89 LPLGVLMKG 386 PRAME 80-89 CLPLGVLMKG 387 PRAME 76-85 WPFTCLPLGV 388 PRAME 51-59 ELFPPLFMA 389 PRAME 49-57 PRELFPPLF 390 PRAME 48-57 LPRELFPPLF 391 PRAME 50-58 RELFPPLFM 392 PRAME 49-58 PRELFPPLFM 393 PSA 239-246 RPSLYTKV 394 PSA 238-246 ERPSLYTKV 395 PSA 236-243 LPERPSLY 396 PSA 235-243 ALPERPSLY 397 PSA 241-249 SLYTKVVHY 398 PSA 240-249 PSLYTKVVHY 399 PSA 239-247 RPSLYTKVV 400 PSMA 211-218 GNKVKNAQ 401 PSMA 202-209 IARYGKVF 402 PSMA 217-225 AQLAGAKGV 403 PSMA 207-215 KVFRGNKVK 404 PSMA 211-219 GNKVKNAQL 405 PSMA 269-277 TPGYPANEY 406 PSMA 268-277 LTPGYPANEY 407 PSMA 271-279 GYPANEYAY 408 PSMA 270-279 PGYPANEYAY 409 PSMA 266-274 DPLTPGYPA 410 PSMA 492-500 SLYESWTKK 411 PSMA 491-500 KSLYESWTKK 412 PSMA 486-494 EGFEGKSLY 413 PSMA 485-494 DEGFEGKSLY 414 PSMA 498-506 TKKSPSPEF 415 PSMA 497-506 WTKKSPSPEF 416 PSMA 492-501 SLYESWTKKS 417 PSMA 725-732 WGEVKRQI 418 PSMA 724-732 AWGEVKRQI 419 PSMA 723-732 KAWGEVKRQI 420 PSMA 723-730 KAWGEVKR 421 PSMA 722-730 SKAWGEVKR 422 PSMA 731-739 QIYVAAIFTV 423 PSMA 733-741 YVAAFTVQA 424 PSMA 725-733 WGEVKRQIY 425 PSMA 727-735 EVKRQ1YVA 426 PSMA 738-746 TVQAAAETL 427 PSMA 737-746 FTVQAAAETL 428 PSMA 729-737 KRQIYVAAF 429 PSMA 721-729 PSKAWGEVK 430 PSMA 723-731 KAWGEVKRQ 431 PSMA 100-108 WKEFGLDSV 432 PSMA 99-108 QWKEFGLDSV 433 PSMA 102-111 EFGLDSVELA 434 SCP-1 126-134 ELRQKESKL 435 SCP-1 125-134 AELRQKESKL 436 SCP-1 133-141 KLQENRKII 437 SCP-1 298-305 QLEEKTKL 438 SCP-1 297-305 NQLEEKTKL 439 SCP-1 288-296 LLEESRDKV 440 SCP-1 287-296 FLLEESRDKV 441 SCP-1 291-299 ESRDKVNQL 442 SCP-1 290-299 EESRDKVNQL 443 SCP-1 475-483 EKBVHIDLEY 444 SCP-1 474-483 RFKEVHDLEY 445 SCP-1 480-488 DLEYSYCIIY 446 SCP-1 477-485 EVHDLEYSY 447 SCP-1 477-486 EVUDLEYSYC 448 SCP-1 502-509 KLSSKREL 449 SCP-1 508-515 ELKNTEYF 450 SCP-1 507-515 RELKNTEYF 451 SCP-1 496-503 KRGQRPKL 452 SCP-1 494-503 LPKRGQRPKL 453 SCP-1 509-517 LKNTEYFTL 454 SCP-1 508-517 ELKNTEYFTL 455 SCP-1 506-514 KRELKNTEY 456 SCP-1 502-510 KLSSKRELK 457 SCP-1 498-506 GQRPKLSSK 458 SCP-1 497-506 RGQRPKLSSK 459 SCP-1 500-508 RPKLSSKRE 460 SCP-1 573-580 LEYVREEL 461 SCP-1 572-580 ELEYVREEL 462 SCP-1 571-580 NELEYVREEL 463 SCP-1 579-587 ELKQKREDEV 464 SCP-1 575-583 YVREELKQK 465 SCP-1 632-640 QLNVYEIIKV 466 SCP-1 630-638 SKQLNVYEI 467 SCP-1 628-636 AESKQLNVY 468 SCP-1 627-636 TAESKQLNYY 469 SCP-1 638-645 JKVNKLEL 470 SCP-1 637-645 EIIKVNKLEL 471 SCP-1 636-645 YEIKVNKLEL 472 SCP-1 642-650 KLELELESA 473 SCP-1 635-643 VYETKVNIKL 474 SCP-1 634-643 NVYETKVNKL 475 SCP-1 646-654 ELESAKQKF 476 SCP-1 642-650 KLELELESA 477 SCP-1 646-654 ELESAKiQKF 478 SCP-1 771-778 KEKLKREA 479 SCP-1 777-785 EAKENTATL 480 SCP-1 776-785 REAKENTATL 481 SCP-1 773-782 KLKREAKENT 482 SCP-1 112-119 EAEKTKKW 483 SCP-1 101-109 GLSRVYSKL 484 SCP-1 100-109 EGLSRVYSKL 485 SCP-1 108-116 KLYKEAEKI 486 SCP-1 98-106 NSEGLSRVY 487 SGP-1 97-106 ENSEGLSRVY 488 SCP-1 102-110 LSRVYSKLY 489 SCP-1 101-110 GLSRVYSKLY 490 SCP-1 96-105 LENSEGLSRV 491 SCP-1 108-117 KLYKEAEKIIK 492 SCP-1 949-956 REDRWAVI 493 SCP-1 948-956 MREDRWAVI 494 SCP-1 947-956 KMREDRWAVI 495 SCP-1 947-955 KMREDRWAV 496 SCP-1 934-942 TTPGSTLKF 497 SGP-1 933-942 LTTPGSTLKF 498 SCP-1 937-945 GSTLKGAI 499 SCP-1 945-953 IRKMREDRW 500 SCP-1 236-243 RLEMIIFKL 501 SCP-1 235-243 SRLEMHFKL 502 SCP-1 242-250 KLKEDYEKI 503 SCP-1 249-257 KIQHLEQEY 504 SCP-1 248-257 EKIQHLEQEY 505 SCP-1 233-242 ENSRLEMHF 506 SCP-1 236-245 RLEMHIFKLKE 507 SCP-1 324-331 LEDIKVSL 508 SCP-1 323-331 ELEDIKVSL 509 SCP-1 322-331 KELEDIKVSL 510 SCP-1 320-327 LTKELEDI 511 SCP-1 319-327 HLTKELEDI 512 SCP-1 330-338 SLQRSVSTQ 513 SCP-1 321-329 TKELEDIKV 514 SCP-1 320-329 LTKELEDIKV 515 SCP-1 326-335 DIKVSLQRSV 516 SCP-1 281-288 KMKDLTFL 517 SCP-1 280-288 NKMKDLTFL 518 SCP-1 279-288 ENKMKDLTFL 519 SCP-1 288-296 LLEESRDKV 520 SCP-1 287-296 FLLEESRDKV 521 SCP-1 291-299 ESRDKVNQL 522 SCP-1 290-299 EESRDKVNQL 523 SCP-1 277-285 EKENKMKDL 524 SCP-1 276-285 TEKENKMKDL 525 SCP-1 279-287 ENKMKDLTF 526 SCP-1 218-225 IEKMITAF 527 SCP-1 217-225 NIEKIVHTAF 528 SCP-1 216-225 SNIEKMITAF 529 SCP-1 223-230 TAFEELRV 530 SCP-1 222-230 ITAFEELRV 531 SCP-1 221-230 MITAFEELRV 532 SCP-1 220-228 KMITAFEEL 533 SCP-1 219-228 EKMITAFEEL 534 SCP-1 227-235 ELRVQAENS 535 SCP-1 213-222 DLNSNIEKMI 536 SCP-1 837-844 WTSAKNTL 537 SCP-1 846-854 TPLPKAYTV 538 SCP-1 845-854 STPLPKAYTV 539 SCP-1 844-852 LSTPLPKAY 540 SCP-1 843-852 TLSTPLPKAY 541 SCP-1 842-850 NTLSTPLPK 542 SCP-1 841-850 KNTLSTPLPK 543 SCP-1 828-835 ISKDKRDY 544 SCP-1 826-835 HGISKDKRDY 545 SCP-1 832-840 KRDYLWTSA 546 SCP-1 829-838 SKDKRDYLWT 547 SCP-1 279-286 ENKMKDLT 548 SCP-1 260-268 EINDKEKQV 549 SCP-1 274-282 QITEKENKM 550 SCP-1 269-277 SLLLIQITE 551 SCP-1 453-460 FEKIAEEL 552 SCP-1 452-460 QFEKIAEEL 553 SCP-1 451-460 KQFEKIAEEL 554 SCP-1 449-456 DNKQFEKI 555 SCP-1 448-456 YDNKQFEKI 556 SCP-1 447-456 LYDNKQFEKI 557 SCP-1 440-447 LGEKETLL 558 SCP-1 439-447 VLGEKETLL 559 SCP-1 438-447 KVLGEKETLL 560 SCP-1 390-398 LLRTEQQRL 561 SCP-1 389-398 ELLRTEQQRL 562 SCP-1 393-401 TEQQRLENY 563 SCP-1 392-401 RTEQQRLENY 564 SCP-1 402-410 EDQLIILTM 565 SCP-1 397-406 RLENYEDQLI 566 SCP-1 368-375 KARAAHSF 567 SCP-1 376-384 VVTEFETTV 568 SCP-1 375-384 FVVTEFETTV 569 SCP-1 377-385 VTEFETTVC 570 SCP-1 376-385 VVTEFETTVC 571 SCP-1 344-352 DLQIATNTI 572 SCP-1 347-355 IATNTICQL 573 SCP-1 346-355 QIATNTICQL 574 SSX4 57-65 VMTKLGFKY 575 SSX4 53-61 LNYEVMTKL 576 SSX4 52-61 KLNYEVMTKL 577 SSX4 66-74 TLPPFMRSK 578 SSX4 110-118 KTMPKKPAE 579 SSX4 103-112 SLQRTFPKJM 580 Tyr 463-471 YIKSYLEQA 581 Tyr 459-467 SFQDYIKSY 582 Tyr 458-467 DSFQDYIKSY 583 Tyr 507-514 LPEEKQPL 584 Tyr 506-514 QLPEEKQPL 585 Tyr 505-514 KQLPEEKQPL 586 Tyr 507-515 LPEEKQPLL 587 Tyr 506-515 QLPEEKQPLL 588 Tyr 497-505 SLLCRHKRK 589 ED-B domain of EVIPQLTDLSFVDITDSSIGLRWTPLN Fibronectin SSTIIGYRITVVAAGEGIPIFEDFVDS SVGYYTVTGLEPGIDYDISVITLINGG ESAPTTLTQQT 590 ED-B domain of CTFDNLSPGLEYNVSVYTVKDDKESVP Fibronectin with ISDTIIPEVPQLTDLSFVDITDSSIGL flanking sequence RWTPLNSSTIIGYRITVVAAGEGIIPI from Fribronectin FEDFVDSSVGYYTVTGLEPGIDYDISV ITLINGGESAPTTLTQQTAVPPPTDLR FTNIGPDTMRVTW 591 ED-B domain of Accession number: X07717 Fibronectin cds 592 CEA protein Accession number: P06731 593 CEA cDNA Accession number: NM_004363 594 Her2/Neu protein Accession number: P04626 595 Her2/Neu cDNA Accession number: M11730 596 SCP-1 protein Accession number: Q15431 597 SCP-1 cDNA Accession number: X95654 598 SSX-4 protein Accession number: 060224 599 SSX-4 cDNA Accession number: NM_005636 *Any of SEQ ID NOS. 1, 8, 9, 11-23, 26-29, 32-44, 47-54, 56-63, 66-68 88-253, and 256-588 can be useful as epitopes in any of the various embodiments of the invention. Any of SEQ ID NOS. 10, 30, 31, 45, 46, 55, 64, 65, 69, 254, and 255 can be useful as sequences containing epitopes or epitope clusters, as described in various embodiments of the invention. **All accession numbers used here and throughout can be accessed through the NCBI databases, for example, through the Entrez seek and retrieval system on the world wide web. Note that the following discussion sets forth the inventors' understanding of the operation of the invention. However, it is not intended that this discussion limit the patent to any particular theory of operation not set forth in the claims.
[0100] In pursuing the development of epitope vaccines others have generated lists of predicted epitopes based on MHC binding motifs. Such peptides can be immunogenic, but may not correspond to any naturally produced antigenic fragment. Therefore, whole antigen will not elicit a similar response or sensitize a target cell to cytolysis by CTL. Therefore such lists do not differentiate between those sequences that can be useful as vaccines and those that cannot. Efforts to determine which of these predicted epitopes are in fact naturally produced have often relied on screening their reactivity with tumor infiltrating lymphocytes (TIL). However, TIL are strongly biased to recognize immune epitopes whereas tumors (and chronically infected cells) will generally present housekeeping epitopes. Thus, unless the epitope is produced by both the housekeeping and immunoproteasomes, the target cell will generally not be recognized by CTL induced with TIL-identified epitopes. The epitopes of the present invention, in contrast, are generated by the action of a specified proteasome, indicating that they can be naturally produced, and enabling their appropriate use. The importance of the distinction between housekeeping and immune epitopes to vaccine design is more fully set forth in PCT publication WO 01/82963 A, which is hereby incorporated by reference in its entirety.
[0101] The epitopes of the invention include or encode polypeptide fragments of TAAs that are precursors or products of proteasomal cleavage by a housekeeping or immune proteasome, and that contain or consist of a sequence having a known or predicted affinity for at least one allele of MHC I. In some embodiments, the epitopes include or encode a polypeptide of about 6 to 25 amino acids in length, preferably about 7 to 20 amino acids in length, more preferably about 8 to 15 amino acids in length, and still more preferably 9 or 10 amino acids in length. However, it is understood that the polypeptides can be larger as long as N-terminal trimming can produce the MHC epitope or that they do not contain sequences that cause the polypeptides to be directed away from the proteasome or to be destroyed by the proteasome. For immune epitopes, if the larger peptides do not contain such sequences, they can be processed in the pAPC by the immune proteasome. Housekeeping epitopes may also be embedded in longer sequences provided that the sequence is adapted to facilitate liberation of the epitope's C-terminus by action of the immunoproteasome. The foregoing discussion has assumed that processing of longer epitopes proceeds through action of the immunoproteasome of the pAPC. However, processing can also be accomplished through the contrivance of some other mechanism, such as providing an exogenous protease activity and a sequence adapted so that action of the protease liberates the MHC epitope. The sequences of these epitopes can be subjected to computer analysis in order to calculate physical, biochemical, immunologic, or molecular genetic properties such as mass, isoelectric point, predicted mobility in electrophoresis, predicted binding to other MHC molecules, melting temperature of nucleic acid probes, reverse translations, similarity or homology to other sequences, and the like.
[0102] In constructing the polynucleotides encoding the polypeptide epitopes of the invention, the gene sequence of the associated TAA can be used, or the polynucleotide can be assembled from any of the corresponding codons. For a 10 amino acid epitope this can constitute on the order of 106 different sequences, depending on the particular amino acid composition. While large, this is a distinct and readily definable set representing a miniscule fraction of the >1018 possible polynucleotides of this length, and thus in some embodiments, equivalents of a particular sequence disclosed herein encompass such distinct and readily definable variations on the listed sequence. In choosing a particular one of these sequences to use in a vaccine, considerations such as codon usage, self-complementarity, restriction sites, chemical stability, etc. can be used as will be apparent to one skilled in the art.
[0103] The invention contemplates producing peptide epitopes. Specifically these epitopes are derived from the sequence of a TAA, and have known or predicted affinity for at least one allele of MHC I. Such epitopes are typically identical to those produced on target cells or pAPCs.
[0104] Compositions Containing Active Epitopes
[0105] Embodiments of the present invention provide polypeptide compositions, including vaccines, therapeutics, diagnostics, pharmacological and pharmaceutical compositions. The various compositions include newly identified epitopes of TAAs, as well as variants of these epitopes. Other embodiments of the invention provide polynucleotides encoding the polypeptide epitopes of the invention. The invention further provides vectors for expression of the polypeptide epitopes for purification. In addition, the invention provides vectors for the expression of the polypeptide epitopes in an APC for use as an anti-tumor vaccine. Any of the epitopes or antigens, or nucleic acids encoding the same, from Table 1 can be used. Other embodiments relate to methods of making and using the various compositions.
[0106] A general architecture for a class I MHC-binding epitope can be described, and has been reviewed more extensively in Madden, D. R. Annu. Rev. Immunol. 13:587-622, 1995, which is hereby incorporated by reference in its entirety. Much of the binding energy arises from main chain contacts between conserved residues in the MHC molecule and the N- and C-termini of the peptide. Additional main chain contacts are made but vary among MHC alleles. Sequence specificity is conferred by side chain contacts of so-called anchor residues with pockets that, again, vary among MHC alleles. Anchor residues can be divided into primary and secondary. Primary anchor positions exhibit strong preferences for relatively well-defined sets of amino acid residues. Secondary positions show weaker and/or less well-defined preferences that can often be better described in terms of less favored, rather than more favored, residues. Additionally, residues in some secondary anchor positions are not always positioned to contact the pocket on the MHC molecule at all. Thus, a subset of peptides exists that bind to a particular MHC molecule and have a side chain-pocket contact at the position in question and another subset exists that show binding to the same MHC molecule that does not depend on the conformation the peptide assumes in the peptide-binding groove of the MHC molecule. The C-terminal residue (P; omega) is preferably a primary anchor residue. For many of the better studied HLA molecules (e.g. A2, A68, B27, B7, B35, and B53) the second position (P2) is also an anchor residue. However, central anchor residues have also been observed including P3 and P5 in HLA-B8, as well as P5 and P (omega)-3 in the murine MHC molecules H-2Db and H-2 Kb, respectively. Since more stable binding will generally improve immunogenicity, anchor residues are preferably conserved or optimized in the design of variants, regardless of their position.
[0107] Because the anchor residues are generally located near the ends of the epitope, the peptide can buckle upward out of the peptide-binding groove allowing some variation in length. Epitopes ranging from 8-11 amino acids have been found for HLA-A68, and up to 13 amino acids for HLA-A2. In addition to length variation between the anchor positions, single residue truncations and extensions have been reported and the N- and C-termini, respectively. Of the non-anchor residues, some point up out of the groove, making no contact with the MHC molecule but being available to contact the TCR, very often P1, P4, and P (omega)-1 for HLA-A2. Others of the non-anchor residues can become interposed between the upper edges of the peptide-binding groove and the TCR, contacting both. The exact positioning of these side chain residues, and thus their effects on binding, MHC fine conformation, and ultimately immunogenicity, are highly sequence dependent. For an epitope to be highly immunogenic it must not only promote stable enough TCR binding for activation to occur, but the TCR must also have a high enough off-rate that multiple TCR molecules can interact sequentially with the same peptide-MHC complex (Kalergis, A. M. et al., Nature Immunol. 2:229-234, 2001, which is hereby incorporated by reference in its entirety). Thus, without further information about the ternary complex, both conservative and non-conservative substitutions at these positions merit consideration when designing variants.
[0108] The polypeptide epitope variants can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations. Variants can be derived from substitution, deletion or insertion of one or more amino acids as compared with the native sequence. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a threonine with a serine, for example. Such replacements are referred to as conservative amino acid replacements, and all appropriate conservative amino acid replacements are considered to be embodiments of one invention. Insertions or deletions can optionally be in the range of about 1 to 4, preferably 1 to 2, amino acids. It is generally preferable to maintain the “anchor positions” of the peptide which are responsible for binding to the MHC molecule in question. Indeed, immunogenicity of peptides can be improved in many cases by substituting more preferred residues at the anchor positions (Franco, et al., Nature Immunology, 1(2):145-150, 2000, which is hereby incorporated by reference in its entirety). Immunogenicity of a peptide can also often be improved by substituting bulkier amino acids for small amino acids found in non-anchor positions while maintaining sufficient cross-reactivity with the original epitope to constitute a useful vaccine. The variation allowed can be determined by routine insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the polypeptide epitope. Because the polypeptide epitope is often 9 amino acids, the substitutions preferably are made to the shortest active epitope, for example, an epitope of 9 amino acids.
[0109] Variants can also be made by adding any sequence onto the N-terminus of the polypeptide epitope variant. Such N-terminal additions can be from 1 amino acid up to at least 25 amino acids. Because peptide epitopes are often trimmed by N-terminal exopeptidases active in the pAPC, it is understood that variations in the added sequence can have no effect on the activity of the epitope. In preferred embodiments, the amino acid residues between the last upstream proteasomal cleavage site and the N-terminus of the MHC epitope do not include a proline residue. Serwold, T. at al., Nature Immunol. 2:644-651, 2001, which is hereby incorporated by reference in its entirety. Accordingly, effective epitopes can be generated from precursors larger than the preferred 9-mer class I motif.
[0110] Generally, peptides are useful to the extent that they correspond to epitopes actually displayed by MHC I on the surface of a target cell or a pACP. A single peptide can have varying affinities for different MHC molecules, binding some well, others adequately, and still others not appreciably (Table 2). MHC alleles have traditionally been grouped according to serologic reactivity which does not reflect the structure of the peptide-binding groove, which can differ among different alleles of the same type. Similarly, binding properties can be shared across types; groups based on shared binding properties have been termed supertypes. There are numerous alleles of MHC I in the human population; epitopes specific to certain alleles can be selected based on the genotype of the patient. 4 TABLE 2 Predicted Binding of Tyrosinase207-216 (SEQ ID NO. 1) to Various MHC types *Half time of MHC I type dissociation (min) A1 0.05 A*0201 1311. A*0205 50.4 A3 2.7 A*1101 0.012 (part of the A3 supertype) A24 6.0 B7 4.0 B8 8.0 B14 60.0 (part of the B27 supertype) B*2702 0.9 B*2705 30.0 B*3501 2.0 (part of the B7 supertype) B*4403 0.1 B*5101 26.0 (part of the B7 supertype) B*5102 55.0 B*5801 0.20 B60 0.40 B62 2.0 *HLA Peptide Binding Predictions (world wide web hypertext transfer protocol “access at bimas.dcrt.nih.gov/molbio/hla_bin”).
[0111] In further embodiments of the invention, the epitope, as peptide or encoding polynucleotide, can be administered as a pharmaceutical composition, such as, for example, a vaccine or an immunogenic composition, alone or in combination with various adjuvants, carriers, or excipients. It should be noted that although the term vaccine may be used throughout the discussion herein, the concepts can be applied and used with any other pharmaceutical composition, including those mentioned herein. Particularly advantageous adjuvants include various cytokines and oligonucleotides containing immunostimulatory sequences (as set forth in greater detail in the co-pending applications referenced herein). Additionally the polynucleotide encoded epitope can be contained in a virus (e.g. vaccinia or adenovirus) or in a microbial host cell (e.g. Salmonella or Listeria monocytogenes) which is then used as a vector for the polynucleotide (Dietrich, G. et al. Nat. Biotech. 16:181-185, 1998, which is hereby incorporated by reference in its entirety). Alternatively a pAPC can be transformed, ex vivo, to express the epitope, or pulsed with peptide epitope, to be itself administered as a vaccine. To increase efficiency of these processes, the encoded epitope can be carried by a viral or bacterial vector, or complexed with a ligand of a receptor found on pAPC. Similarly the peptide epitope can be complexed with or conjugated to a pAPC ligand. A vaccine can be composed of more than a single epitope.
[0112] Particularly advantageous strategies for incorporating epitopes and/or epitope clusters, into a vaccine or pharmaceutical composition are disclosed in U.S. patent application Ser. No. 09/560,465 entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” filed on Apr. 28, 2000, which is hereby incorporated by reference in its entirety. Epitope clusters for use in connection with this invention are disclosed in U.S. patent application Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,” filed on Apr. 28, 2000, which is hereby incorporated by reference in its entirety.
[0113] Preferred embodiments of the present invention are directed to vaccines and methods for causing a pAPC or population of pAPCs to present housekeeping epitopes that correspond to the epitopes displayed on a particular target cell. Any of the epitopes or antigens in Table 1, can be used for example. In one embodiment, the housekeeping epitope is a TuAA epitope processed by the housekeeping proteasome of a particular tumor type. In another embodiment, the housekeeping epitope is a virus-associated epitope processed by the housekeeping proteasome of a cell infected with a virus. This facilitates a specific T cell response to the target cells. Concurrent expression by the pAPCs of multiple epitopes, corresponding to different induction states (pre- and post-attack), can drive a CTL response effective against target cells as they display either housekeeping epitopes or immune epitopes.
[0114] By having both housekeeping and immune epitopes present on the pAPC, this embodiment can optimize the cytotoxic T cell response to a target cell. With dual epitope expression, the pAPCs can continue to sustain a CTL response to the immune-type epitope when the tumor cell switches from the housekeeping proteasome to the immune proteasome with induction by IFN, which, for example, may be produced by tumor-infiltrating CTLs.
[0115] In a preferred embodiment, immunization of a patient is with a vaccine that includes a housekeeping epitope. Many preferred TAAs are associated exclusively with a target cell, particularly in the case of infected cells. In another embodiment, many preferred TAAs are the result of deregulated gene expression in transformed cells, but are found also in tissues of the testis, ovaries and fetus. In another embodiment, useful TAAs are expressed at higher levels in the target cell than in other cells. In still other embodiments, TAAs are not differentially expressed in the target cell compare to other cells, but are still useful since they are involved in a particular function of the cell and differentiate the target cell from most other peripheral cells; in such embodiments, healthy cells also displaying the TAA may be collaterally attacked by the induced T cell response, but such collateral damage is considered to be far preferable to the condition caused by the target cell.
[0116] The vaccine contains a housekeeping epitope in a concentration effective to cause a pAPC or populations of pAPCs to display housekeeping epitopes. Advantageously, the vaccine can include a plurality of housekeeping epitopes or one or more housekeeping epitopes optionally in combination with one or more immune epitopes. Formulations of the vaccine contain peptides and/or nucleic acids in a concentration sufficient to cause pAPCs to present the epitopes. The formulations preferably contain epitopes in a total concentration of about 1 &mgr;g-1 mg/100 &mgr;l of vaccine preparation. Conventional dosages and dosing for peptide vaccines and/or nucleic acid vaccines can be used with the present invention, and such dosing regimens are well understood in the art. In one embodiment, a single dosage for an adult human may advantageously be from about 1 to about 5000 &mgr;l of such a composition, administered one time or multiple times, e.g., in 2, 3, 4 or more dosages separated by 1 week, 2 weeks, 1 month, or more. insulin pump delivers 1 ul per hour (lowest frequency) ref intranodal method patent.
[0117] The compositions and methods of the invention disclosed herein further contemplate incorporating adjuvants into the formulations in order to enhance the performance of the vaccines. Specifically, the addition of adjuvants to the formulations is designed to enhance the delivery or uptake of the epitopes by the pAPCs. The adjuvants contemplated by the present invention are known by those of skill in the art and include, for example, GMCSF, GCSF, IL-2, IL-12, BCG, tetanus toxoid, osteopontin, and ETA-1.
[0118] In some embodiments of the invention, the vaccines can include a recombinant organism, such as a virus, bacterium or parasite, genetically engineered to express an epitope in a host. For example, Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting TuAAs to the immune system. In a preferred embodiment, this vector can be engineered to express a housekeeping epitope to induce therapeutic responses. The normal route of infection of this organism is through the gut and can be delivered orally. In another embodiment, an adenovirus (Ad) vector encoding a housekeeping epitope for a TuAA can be used to induce anti-virus or anti-tumor responses. Bone marrow-derived dendritic cells can be transduced with the virus construct and then injected, or the virus can be delivered directly via subcutaneous injection into an animal to induce potent T-cell responses. Another embodiment employs a recombinant vaccinia virus engineered to encode amino acid sequences corresponding to a housekeeping epitope for a TAA. Vaccinia viruses carrying constructs with the appropriate nucleotide substitutions in the form of a minigene construct can direct the expression of a housekeeping epitope, leading to a therapeutic T cell response against the epitope.
[0119] The immunization with DNA requires that APCs take up the DNA and express the encoded proteins or peptides. It is possible to encode a discrete class I peptide on the DNA. By immunizing with this construct, APCs can be caused to express a housekeeping epitope, which is then displayed on class I MHC on the surface of the cell for stimulating an appropriate CTL response. Constructs generally relying on termination of translation or non-proteasomal proteases for generation of proper termini of housekeeping epitopes have been described in U.S. patent application Ser. No. 09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS, filed on Apr. 28, 2000.
[0120] As mentioned, it can be desirable to express housekeeping peptides in the context of a larger protein. Processing can be detected even when a small number of amino acids are present beyond the terminus of an epitope. Small peptide hormones are usually proteolytically processed from longer translation products, often in the size range of approximately 60-120 amino acids. This fact has led some to assume that this is the minimum size that can be efficiently translated. In some embodiments, the housekeeping peptide can be embedded in a translation product of at least about 60 amino acids. In other embodiments the housekeeping peptide can be embedded in a translation product of at least about 50, 30, or 15 amino acids.
[0121] Due to differential proteasomal processing, the immune proteasome of the pAPC produces peptides that are different from those produced by the housekeeping proteasome in peripheral body cells. Thus, in expressing a housekeeping peptide in the context of a larger protein, it is preferably expressed in the APC in a context other than its full length native sequence, because, as a housekeeping epitope, it is generally only efficiently processed from the native protein by the housekeeping proteasome, which is not active in the APC. In order to encode the housekeeping epitope in a DNA sequence encoding a larger protein, it is useful to find flanking areas on either side of the sequence encoding the epitope that permit appropriate cleavage by the immune proteasome in order to liberate that housekeeping epitope. Altering flanking amino acid residues at the N-terminus and C-terminus of the desired housekeeping epitope can facilitate appropriate cleavage and generation of the housekeeping epitope in the APC. Sequences embedding housekeeping epitopes can be designed de novo and screened to determine which can be successfully processed by immune proteasomes to liberate housekeeping epitopes.
[0122] Alternatively, another strategy is very effective for identifying sequences allowing production of housekeeping epitopes in APC. A contiguous sequence of amino acids can be generated from head to tail arrangement of one or more housekeeping epitopes. A construct expressing this sequence is used to immunize an animal, and the resulting T cell response is evaluated to determine its specificity to one or more of the epitopes in the array. By definition, these immune responses indicate housekeeping epitopes that are processed in the pAPC effectively. The necessary flanking areas around this epitope are thereby defined. The use of flanking regions of about 4-6 amino acids on either side of the desired peptide can provide the necessary information to facilitate proteasome processing of the housekeeping epitope by the immune proteasome. Therefore, a sequence ensuring epitope synchronization of approximately 16-22 amino acids can be inserted into, or fused to, any protein sequence effectively to result in that housekeeping epitope being produced in an APC. In alternate embodiments the whole head-to-tail array of epitopes, or just the epitopes immediately adjacent to the correctly processed housekeeping epitope can be similarly transferred from a test construct to a vaccine vector.
[0123] In a preferred embodiment, the housekeeping epitopes can be embedded between known immune epitopes, or segments of such, thereby providing an appropriate context for processing. The abutment of housekeeping and immune epitopes can generate the necessary context to enable the immune proteasome to liberate the housekeeping epitope, or a larger fragment, preferably including a correct C-terminus. It can be useful to screen constructs to verify that the desired epitope is produced. The abutment of housekeeping epitopes can generate a site cleavable by the immune proteasome. Some embodiments of the invention employ known epitopes to flank housekeeping epitopes in test substrates; in others, screening as described below are used whether the flanking regions are arbitrary sequences or mutants of the natural flanking sequence, and whether or not knowledge of proteasomal cleavage preferences are used in designing the substrates.
[0124] Cleavage at the mature N-terminus of the epitope, while advantageous, is not required, since a variety of N-terminal trimming activities exist in the cell that can generate the mature N-terminus of the epitope subsequent to proteasomal processing. It is preferred that such N-terminal extension be less than about 25 amino acids in length and it is further preferred that the extension have few or no proline residues. Preferably, in screening, consideration is given not only to cleavage at the ends of the epitope (or at least at its C-terminus), but consideration also can be given to ensure limited cleavage within the epitope.
[0125] Shotgun approaches can be used in designing test substrates and can increase the efficiency of screening. In one embodiment multiple epitopes can be assembled one after the other, with individual epitopes possibly appearing more than once. The substrate can be screened to determine which epitopes can be produced. In the case where a particular epitope is of concern a substrate can be designed in which it appears in multiple different contexts. When a single epitope appearing in more than one context is liberated from the substrate additional secondary test substrates, in which individual instances of the epitope are removed, disabled, or are unique, can be used to determine which are being liberated and truly constitute sequences ensuring epitope synchronization.
[0126] Several readily practicable screens exist. A preferred in vitro screen utilizes proteasomal digestion analysis, using purified immune proteasomes, to determine if the desired housekeeping epitope can be liberated from a synthetic peptide embodying the sequence in question. The position of the cleavages obtained can be determined by techniques such as mass spectrometry, HPLC, and N-terminal pool sequencing; as described in greater detail in U.S. Patent Applications entitled METHOD OF EPITOPE DISCOVERY, EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, two Provisional U.S. Patent Applications entitled EPITOPE SEQUENCES, which are all cited and incorporated by reference above.
[0127] Alternatively, in vivo screens such as immunization or target sensitization can be employed. For immunization a nucleic acid construct capable of expressing the sequence in question is used. Harvested CTL can be tested for their ability to recognize target cells presenting the housekeeping epitope in question. Such targets cells are most readily obtained by pulsing cells expressing the appropriate MHC molecule with synthetic peptide embodying the mature housekeeping epitope. Alternatively, cells known to express housekeeping proteasome and the antigen from which the housekeeping epitope is derived, either endogenously or through genetic engineering, can be used. To use target sensitization as a screen, CTL, or preferably a CTL clone, that recognizes the housekeeping epitope can be used. In this case it is the target cell that expresses the embedded housekeeping epitope (instead of the pAPC during immunization) and it must express immune proteasome. Generally, the target cell can be transformed with an appropriate nucleic acid construct to confer expression of the embedded housekeeping epitope. Loading with a synthetic peptide embodying the embedded epitope using peptide loaded liposomes or a protein transfer reagent such as BIOPORTER™ (Gene Therapy Systems, San Diego, Calif.) represents an alternative.
[0128] Additional guidance on nucleic acid constructs useful as vaccines in accordance with the present invention are disclosed in U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed on Apr. 28, 2000. Further, expression vectors and methods for their design, which are useful in accordance with the present invention are disclosed in U.S. Patent Application No. 60/336,968 (attorney docket number CTLIMM.022PR) entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN,” filed on Nov. 7, 2001, which is incorporated by reference in its entirety.
[0129] A preferred embodiment of the present invention includes a method of administering a vaccine including an epitope (or epitopes) to induce a therapeutic immune response. The vaccine is administered to a patient in a manner consistent with the standard vaccine delivery protocols that are known in the art. Methods of administering epitopes of TAAs including, without limitation, transdermal, intranodal, perinodal, oral, intravenous, intradermal, intramuscular, intraperitoneal, and mucosal administration, including delivery by injection, instillation or inhalation. A particularly useful method of vaccine delivery to elicit a CTL response is disclosed in Australian Patent No. 739189 issued Jan. 17, 2002; U.S. patent application Ser. No. 09/380,534, filed on Sep. 1, 1999; and a Continuation-in-Part thereof U.S. patent application Ser. No. 09/776,232 both entitled “A METHOD OF INDUCING A CTL RESPONSE,” filed on Feb. 2, 2001.
[0130] Reagents Recognizing Epitopes
[0131] In another aspect of the invention, proteins with binding specificity for the epitope and/or the epitope-MHC molecule complex are contemplated, as well as the isolated cells by which they can be expressed. In one set of embodiments these reagents take the form of immunoglobulins: polyclonal sera or monoclonal antibodies (mAb), methods for the generation of which are well know in the art. Generation of mAb with specificity for peptide-MHC molecule complexes is known in the art. See, for example, Aharoni et al. Nature 351:147-150, 1991; Andersen et al. Proc. Natl. Acad. Sci. USA 93:1820-1824, 1996; Dadaglio et al. Immunity 6:727-738, 1997; Duc et al. Int. Immunol. 5:427-431,1993; Eastman et al. Eur. J. Immunol. 26:385-393, 1996; Engberg et al. Immunotechnology 4:273-278, 1999; Porgdor et al. Immunity 6:715-726, 1997; Puri et al. J. Immunol. 158:2471-2476, 1997; and Polakova, K., et al. J. Immunol. 165 342-348, 2000; all of which are hereby incorporated by reference in their entirety.
[0132] In other embodiments the compositions can be used to induce and generate, in vivo and in vitro, T-cells specific for the any of the epitopes and/or epitope-MHC complexes. In preferred embodiments the epitope can be any one or more of those listed in TABLE 1, for example. Thus, embodiments also relate to and include isolated T cells, T cell clones, T cell hybridomas, or a protein containing the T cell receptor (TCR) binding domain derived from the cloned gene, as well as a recombinant cell expressing such a protein. Such TCR derived proteins can be simply the extra-cellular domains of the TCR, or a fusion with portions of another protein to confer a desired property or function. One example of such a fusion is the attachment of TCR binding domains to the constant regions of an antibody molecule so as to create a divalent molecule. The construction and activity of molecules following this general pattern have been reported, for example, Plaksin, D. et al. J. Immunol. 158:2218-2227, 1997 and Lebowitz, M. S. et al. Cell Immunol. 192:175-184, 1999, which are hereby incorporated by reference in their entirety. The more general construction and use of such molecules is also treated in U.S. Pat. No. 5,830,755 entitled T CELL RECEPTORS AND THEIR USE IN THERAPEUTIC AND DIAGNOSTIC METHODS, which is hereby incorporated by reference in its entirety.
[0133] The generation of such T cells can be readily accomplished by standard immunization of laboratory animals, and reactivity to human target cells can be obtained by immunizing with human target cells or by immunizing HLA-transgenic animals with the antigen/epitope. For some therapeutic approaches T cells derived from the same species are desirable. While such a cell can be created by cloning, for example, a murine TCR into a human T cell as contemplated above, in vitro immunization of human cells offers a potentially faster option. Techniques for in vitro immunization, even using naive donors, are know in the field, for example, Stauss et al., Proc. Natl. Acad. Sci. USA 89:7871-7875, 1992; Salgaller et al. Cancer Res. 55:4972-4979, 1995; Tsai et al., J. Immunol. 158:1796-1802, 1997; and Chung et al., J. Immunother. 22:279-287, 1999; which are hereby incorporated by reference in their entirety.
[0134] Any of these molecules can be conjugated to enzymes, radiochemicals, fluorescent tags, and toxins, so as to be used in the diagnosis (imaging or other detection), monitoring, and treatment of the pathogenic condition associated with the epitope. Thus a toxin conjugate can be administered to kill tumor cells, radiolabeling can facilitate imaging of epitope positive tumor, an enzyme conjugate can be used in an ELISA-like assay to diagnose cancer and confirm epitope expression in biopsied tissue. In a further embodiment, such T cells as set forth above, following expansion accomplished through stimulation with the epitope and/or cytokines, can be administered to a patient as an adoptive immunotherapy.
[0135] Reagents Comprising Epitopes
[0136] A further aspect of the invention provides isolated epitope-MHC complexes. In a particularly advantageous embodiment of this aspect of the invention, the complexes can be soluble, multimeric proteins such as those described in U.S. Pat. No. 5,635,363 (tetramers) or U.S. Pat. No. 6,015,884 (Ig-dimers), both of which are hereby incorporated by reference in their entirety. Such reagents are useful in detecting and monitoring specific T cell responses, and in purifying such T cells.
[0137] Isolated MHC molecules complexed with epitopic peptides can also be incorporated into planar lipid bilayers or liposomes. Such compositions can be used to stimulate T cells in vitro or, in the case of liposomes, in vivo. Co-stimulatory molecules (e.g. B7, CD40, LFA-3) can be incorporated into the same compositions or, especially for in vitro work, co-stimulation can be provided by anti-co-receptor antibodies (e.g. anti-CD28, anti-CD154, anti-CD2) or cytokines (e.g. IL-2, IL-12). Such stimulation of T cells can constitute vaccination, drive expansion of T cells in vitro for subsequent infusion in an immuotherapy, or constitute a step in an assay of T cell function.
[0138] The epitope, or more directly its complex with an MHC molecule, can be an important constituent of functional assays of antigen-specific T cells at either an activation or readout step or both. Of the many assays of T cell function current in the art (detailed procedures can be found in standard immunological references such as Current Protocols in Immunology 1999 John Wiley & Sons Inc., N.Y., which is hereby incorporated by reference in its entirety) two broad classes can be defined, those that measure the response of a pool of cells and those that measure the response of individual cells. Whereas the former conveys a global measure of the strength of a response, the latter allows determination of the relative frequency of responding cells. Examples of assays measuring global response are cytotoxicity assays, ELISA, and proliferation assays detecting cytokine secretion. Assays measuring the responses of individual cells (or small clones derived from them) include limiting dilution analysis (LDA), ELISPOT, flow cytometric detection of unsecreted cytokine (described in U.S. Pat. No. 5,445,939, entitled “METHOD FOR ASSESSMENT OF THE MONONUCLEAR LEUKOCYTE IMMUNE SYSTEM” and U.S. Patent Nos 5,656,446; and 5,843,689, both entitled “METHOD FOR THE ASSESSMENT OF THE MONONUCLEAR LEUKOCYTE IMMUNE SYSTEM,” reagents for which are sold by Becton, Dickinson & Company under the tradename ‘FASTIMMUNE’, which patents are hereby incorporated by reference in their entirety) and detection of specific TCR with tetramers or Ig-dimers as stated and referenced above. The comparative virtues of these techniques have been reviewed in Yee, C. et al. Current Opinion in Immunology, 13:141-146, 2001, which is hereby incorporated by reference in its entirety. Additionally detection of a specific TCR rearrangement or expression can be accomplished through a variety of established nucleic acid based techniques, particularly in situ and single-cell PCR techniques, as will be apparent to one of skill in the art.
[0139] These functional assays are used to assess endogenous levels of immunity, response to an immunologic stimulus (e.g. a vaccine), and to monitor immune status through the course of a disease and treatment. Except when measuring endogenous levels of immunity, any of these assays presume a preliminary step of immunization, whether in vivo or in vitro depending on the nature of the issue being addressed. Such immunization can be carried out with the various embodiments of the invention described above or with other forms of immunogen (e.g., pAPC-tumor cell fusions) that can provoke similar immunity. With the exception of PCR and tetramer/Ig-dimer type analyses which can detect expression of the cognate TCR, these assays generally benefit from a step of in vitro antigenic stimulation which can advantageously use various embodiments of the invention as described above in order to detect the particular functional activity (highly cytolytic responses can sometimes be detected directly). Finally, detection of cytolytic activity requires epitope-displaying target cells, which can be generated using various embodiments of the invention. The particular embodiment chosen for any particular step depends on the question to be addressed, ease of use, cost, and the like, but the advantages of one embodiment over another for any particular set of circumstances will be apparent to one of skill in the art.
[0140] The peptide MHC complexes described in this section have traditionally been understood to be non-covalent associations. However it is possible, and can be advantageous, to create a covalent linkages, for example by encoding the epitope and MHC heavy chain or the epitope, &bgr;2-microglobulin, and MHC heavy chain as a single protein (Yu, Y. L. Y., et al., J. Immunol. 168:3145-3149, 2002; Mottez, E., et at., J. Exp. Med. 181:493,1995; Dela Cruz, C. S., et al., Int. Immunol. 12:1293, 2000; Mage, M. G., et al., Proc. Natl. Acad. Sci. USA 89:10658,1992; Toshitani, K., et al., Proc. Natl. Acad. Sci. USA 93:236,1996; Lee, L., et al., Eur. J. Immunol. 24:2633,1994; Chung, D. H., et al., J. Immunol. 163:3699,1999; Uger, R. A. and B. H. Barber, J. Immunol. 160:1598, 1998; Uger, R. A., et al., J. Immunol. 162:6024,1999; and White, J., et al., J. Immunol. 162:2671, 1999; which are incorporated herein by reference in their entirety). Such constructs can have superior stability and overcome roadblocks in the processing-presentation pathway. They can be used in the already described vaccines, reagents, and assays in similar fashion.
[0141] Tumor Associated Antigens
[0142] Epitopes of the present invention are derived from the TuAAs tyrosinase (SEQ ID NO. 2), SSX-2, (SEQ ID NO. 3), PSMA (prostate-specific membrane antigen) (SEQ ID NO. 4), GP100, (SEQ ID NO. 70), MAGE-1, (SEQ ID NO. 71), MAGE-2, (SEQ ID NO. 72), MAGE-3, (SEQ ID NO. 73), NY-ESO-1, (SEQ ID NO. 74), PRAME, (SEQ ID NO. 77), PSA, (SEQ ID NO. 78), PSCA, (SEQ ID NO. 79), the ED-B domain of fibronectin (SEQ ID NOS 589 and 590), CEA (carcinoembryonic antigen) (SEQ ID NO. 592), Her2/Neu (SEQ ID NO. 594), SCP-1 (SEQ ID NO. 596) and SSX-4 (SEQ ID NO. 598). The natural coding sequences for these eleven proteins, or any segments within them, can be determined from their cDNA or complete coding (cds) sequences, SEQ ID NOS. 5-7, 80-87, 591, 593, 595, 597, and 599, respectively.
[0143] Tyrosinase is a melanin biosynthetic enzyme that is considered one of the most specific markers of melanocytic differentiation. Tyrosinase is expressed in few cell types, primarily in melanocytes, and high levels are often found in melanomas. The usefulness of tyrosinase as a TuAA is taught in U.S. Pat. No. 5,747,271 entitled “METHOD FOR IDENTIFYING INDIVIDUALS SUFFERING FROM A CELLULAR ABNORMALITY SOME OF WHOSE ABNORMAL CELLS PRESENT COMPLEXES OF HLA-A2/TYROSINASE DERIVED PEPTIDES, AND METHODS FOR TREATING SAID INDIVIDUALS” which is hereby incorporated by reference in its entirety.
[0144] GP100, also known as PMe117, also is a melanin biosynthetic protein expressed at high levels in melanomas. GP100 as a TuAA is disclosed in U.S. Pat. No. 5,844,075 entitled “MELANOMA ANTIGENS AND THEIR USE IN DIAGNOSTIC AND THERAPEUTIC METHODS,” which is hereby incorporated by reference in its entirety.
[0145] SSX-2, also know as Hom-Mel-40, is a member of a family of highly conserved cancer-testis antigens (Gure, A. O. et al. Int. J. Cancer 72:965-971, 1997, which is hereby incorporated by reference in its entirety). Its identification as a TuAA is taught in U.S. Pat. No. 6,025,191 entitled “ISOLATED NUCLEIC ACID MOLECULES WHICH ENCODE A MELANOMA SPECIFIC ANTIGEN AND USES THEREOF,” which is hereby incorporated by reference in its entirety. Cancer-testis antigens are found in a variety of tumors, but are generally absent from normal adult tissues except testis. Expression of different members of the SSX family have been found variously in tumor cell lines. Due to the high degree of sequence identity among SSX family members, similar epitopes from more than one member of the family will be generated and able to bind to an MHC molecule, so that some vaccines directed against one member of this family can cross-react and be effective against other members of this family (see example 3 below).
[0146] MAGE-1, MAGE-2, and MAGE-3 are members of another family of cancer-testis antigens originally discovered in melanoma (MAGE is a contraction of melanoma-associated antigen) but found in a variety of tumors. The identification of MAGE proteins as TuAAs is taught in U.S. Pat. No. 5,342,774 entitled NUCLEOTIDE SEQUENCE ENCODING THE TUMOR REJECTION ANTIGEN PRECURSOR, MAGE-1, which is hereby incorporated by reference in its entirety, and in numerous subsequent patents. Currently there are 17 entries for (human) MAGE in the SWISS Protein database. There is extensive similarity among these proteins so in many cases, an epitope from one can induce a cross-reactive response to other members of the family. A few of these have not been observed in tumors, most notably MAGE-H1 and MAGE-D1, which are expressed in testes and brain, and bone marrow stromal cells, respectively. The possibility of cross-reactivity on normal tissue is ameliorated by the fact that they are among the least similar to the other MAGE proteins.
[0147] NY-ESO-1, is a cancer-testis antigen found in a wide variety of tumors, also known as CTAG-1 (Cancer-Testis Antigen-1) and CAG-3 (Cancer Antigen-3). NY-ESO-1 as a TuAA is disclosed in U.S. Pat. No. 5,804,381 entitled ISOLATED NUCLEIC ACID MOLECULE ENCODING AN ESOPHAGEAL CANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF, AND USES THEREOF which is hereby incorporated by reference in its entirety. A paralogous locus encoding antigens with extensive sequence identity, LAGE-1a/s (SEQ ID NO. 75) and LAGE-1b/L (SEQ ID NO. 76), have been disclosed in publicly available assemblies of the human genome, and have been concluded to arise through alternate splicing. Additionally, CT-2 (or CTAG-2, Cancer-Testis Antigen-2) appears to be either an allele, a mutant, or a sequencing discrepancy of LAGE-1b/L. Due to the extensive sequence identity, many epitopes from NY-ESO-1 can also induce immunity to tumors expressing these other antigens. See FIG. 1. The proteins are virtually identical through amino acid 70. From 71-134 the longest run of identities between NY-ESO-1 and LAGE is 6 residues, but potentially cross-reactive sequences are present. And from 135-180 NY-ESO and LAGE-1a/s are identical except for a single residue, but LAGE-1b/L is unrelated due to the alternate splice. The CAMEL and LAGE-2 antigens appear to derive from the LAGE-1 mRNA, but from alternate reading frames, thus giving rise to unrelated protein sequences. More recently, GenBank Accession AF277315.5, Homo sapiens chromosome X clone RP5-865E18, RP5-1087L19, complete sequence, reports three independent loci in this region which are labeled as LAGE1 (corresponding to CTAG-2 in the genome assemblies), plus LAGE2-A and LAGE2-B (both corresponding to CTAG-1 in the genome assemblies).
[0148] PSMA (prostate-specific membranes antigen), a TuAA described in U.S. Pat. No. 5,538,866 entitled “PROSTATE-SPECIFIC MEMBRANES ANTIGEN” which is hereby incorporated by reference in its entirety, is expressed by normal prostate epithelium and, at a higher level, in prostatic cancer. It has also been found in the neovasculature of non-prostatic tumors. PSMA can thus form the basis for vaccines directed to both prostate cancer and to the neovasculature of other tumors. This later concept is more fully described in a provisional U.S. Patent application No. 60/274,063 entitled ANTI-NEOVASCULAR VACCINES FOR CANCER, filed Mar. 7, 2001, and U.S. application Ser. No. 10/094,699, attorney docket number CTLIMM.015A, filed on Mar. 7, 2002, entitled “ANTI-NEOVASCULAR PREPARATIONS FOR CANCER,” both of which are hereby incorporated by reference in their entirety. Briefly, as tumors grow they recruit ingrowth of new blood vessels. This is understood to be necessary to sustain growth as the centers of unvascularized tumors are generally necrotic and angiogenesis inhibitors have been reported to cause tumor regression. Such new blood vessels, or neovasculature, express antigens not found in established vessels, and thus can be specifically targeted. By inducing CTL against neovascular antigens the vessels can be disrupted, interrupting the flow of nutrients to (and removal of wastes from) tumors, leading to regression.
[0149] Alternate splicing of the PSMA mRNA also leads to a protein with an apparent start at Met58, thereby deleting the putative membrane anchor region of PSMA as described in U.S. Pat. No. 5,935,818 entitled “ISOLATED NUCLEIC ACID MOLECULE ENCODING ALTERNATIVELY SPLICED PROSTATE-SPECIFIC MEMBRANES ANTIGEN AND USES THEREOF” which is hereby incorporated by reference in its entirety. A protein termed PSMA-like protein, Genbank accession number AF261715, is nearly identical to amino acids 309-750 of PSMA and has a different expression profile. Thus the most preferred epitopes are those with an N-terminus located from amino acid 58 to 308.
[0150] PRAME, also know as MAPE, DAGE, and OIP4, was originally observed as a melanoma antigen. Subsequently, it has been recognized as a CT antigen, but unlike many CT antigens (e.g., MAGE, GAGE, and BAGE) it is expressed in acute myeloid leukemias. PRAME is a member of the MAPE family which consists largely of hypothetical proteins with which it shares limited sequence similarity. The usefulness of PRAME as a TuAA is taught in U.S. Pat. No. 5,830,753 entitled “ISOLATED NUCLEIC ACID MOLECULES CODING FOR TUMOR REJECTION ANTIGEN PRECURSOR DAGE AND USES THEREOF” which is hereby incorporated by reference in its entirety.
[0151] PSA, prostate specific antigen, is a peptidase of the kallikrein family and a differentiation antigen of the prostate. Expression in breast tissue has also been reported. Alternate names include gamma-seminoprotein, kallikrein 3, seminogelase, seminin, and P-30 antigen. PSA has a high degree of sequence identity with the various alternate splicing products prostatic/glandular kallikrein-1and -2, as well as kalilrein 4, which is also expressed in prostate and breast tissue. Other kallikreins generally share less sequence identity and have different expression profiles. Nonetheless, cross-reactivity that might be provoked by any particular epitope, along with the likelihood that that epitope would be liberated by processing in non-target tissues (most generally by the housekeeping proteasome), should be considered in designing a vaccine.
[0152] PSCA, prostate stem cell antigen, and also known as SCAH-2, is a differentiation antigen preferentially expressed in prostate epithelial cells, and overexpresssed in prostate cancers. Lower level expression is seen in some normal tissues including neuroendocrine cells of the digestive tract and collecting ducts of the kidney. PSCA is described in U.S. Pat. No. 5,856,136 entitled “HUMAN STEM CELL ANTIGENS” which is hereby incorporated by reference in its entirety.
[0153] Synaptonemal complex protein 1 (SCP-1), also known as HOM-TES-14, is a meiosis-associated protein and also a cancer-testis antigen (Tureci, O., et al. Proc. Natl. Acad. Sci. USA 95:5211-5216, 1998). As a cancer antigen its expression is not cell-cycle regulated and it is found frequently in gliomas, breast, renal cell, and ovarian carcinomas. It has some similarity to myosins, but with few enough identities that cross-reactive epitopes are not an immediate prospect.
[0154] The ED-B domain of fibronectin is also a potential target. Fibronectin is subject to developmentally regulated alternative splicing, with the ED-B domain being encoded by a single exon that is used primarily in oncofetal tissues (Matsuura, H. and S. Hakomori Proc. Natl. Acad. Sci. USA 82:6517-6521, 1985; Carnemolla, B. et al. J. Cell Biol. 108:1139-1148, 1989; Loridon-Rosa, B. et al. Cancer Res.50:1608-1612, 1990; Nicolo, G. et al. Cell Differ. Dev. 32:401-408, 1990; Borsi, L. et al. Exp. Cell Res. 199:98-105, 1992; Oyama, F. et al. Cancer Res. 53:2005-2011, 1993; Mandel, U. et al. APMIS 102:695-702, 1994; Farnoud, M. R. et al. Int. J. Cancer 61:27-34, 1995; Pujuguet, P. et al. Am. J. Pathol. 148:579-592, 1996; Gabler, U. et al. Heart 75:358-362, 1996;Chevalier, X. Br. J. Rheumatol. 35:407415, 1996; Midulla, M. Cancer Res. 60:164-169, 2000).
[0155] The ED-B domain is also expressed in fibronectin of the neovasculature (Kaczmarek, J. et al. Int. J. Cancer 59:11-16, 1994; Castellani, P. et al. Int. J. Cancer 59:612-618, 1994; Neri, D. et al. Nat. Biotech. 15:1271-1275, 1997; Karelina, T. V. and A. Z. Eisen Cancer Detect. Prev. 22:438-444, 1998; Tarli, L. et al. Blood 94:192-198, 1999; Castellani, P. et al. Acta Neurochir. (Wien) 142:277-282, 2000). As an oncofetal domain, the ED-B domain is commonly found in the fibronectin expressed by neoplastic cells in addition to being expressed by the neovasculature. Thus, CTL-inducing vaccines targeting the ED-B domain can exhibit two mechanisms of action: direct lysis of tumor cells, and disruption of the tumor's blood supply through destruction of the tumor-associated neovasculature. As CTL activity can decay rapidly after withdrawal of vaccine, interference with normal angiogenesis can be minimal. The design and testing of vaccines targeted to neovasculature is described in Provisional U.S. Patent Application No. 60/274,063 entitled “ANTI-NEOVASCULATURE VACCINES FOR CANCER” and in U.S. patent application Ser. No. 10/094,699, attorney docket number CTLIMM.015A, entitled “ANTI-NEOVASCULATURE PREPARATIONS FOR CANCER, filed on date even with this application (Mar. 7, 2002). A tumor cell line is disclosed in Provisional U.S. Application No. 60/363,131, filed on Mar. 7, 2002, attorney docket number CTLIMM.028PR, entitled “HLA-TRANSGENIC MURINE TUMOR CELL LINE,” which is hereby incorporated by reference in its entirety.
[0156] Carcinoembryonic antigen (CEA) is a paradigmatic oncofetal protein first described in 1965 (Gold and Freedman, J. Exp. Med. 121: 439462, 1965. Fuller references can be found in the Online Medelian Inheritance in Man; record * 114890). It has officially been renamed carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). Its expression is most strongly associated with adenocarcinomas of the epithelial lining of the digestive tract and in fetal colon. CEA is a member of the immunoglobulin supergene family and the defining member of the CEA subfamily.
[0157] HER2/NEU is an oncogene related to the epidermal growth factor receptor (van de Vijver, et al., New Eng. J. Med. 319:1239-1245, 1988), and apparently identical to the c-ERBB2 oncogene (Di Fiore, et al., Science 237: 178-182, 1987). The over-expression of ERBB2 has been implicated in the neoplastic transformation of prostate cancer. As HER2 it is amplified and over-expressed in 25-30% of breast cancers among other tumors where expression level is correlated with the aggressiveness of the tumor (Slamon, et al., New Eng. J. Med. 344:783-792, 2001). A more detailed description is available in the Online Medelian Inheritance in Man; record *164870.
[0158] All references mentioned herein are hereby incorporated by reference in their entirety. Further, incorporated by reference in its entirety is U.S. patent application Ser. No. 10/005,905 (attorney docket number CTLIMM.021CP1) entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” filed on Nov. 7, 2001 and a continuation thereof, U.S. Application No. ______, filed on Dec. 7, 2000, attorney docket number CTLIMM.21CP1C, also entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS.”
[0159] Useful epitopes were identified and tested as described in the following examples. However, these examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.
EXAMPLES[0160] Sequences of Specific Preferred Epitopes
Example 1[0161] Manufacture of Epitopes.
[0162] A. Synthetic Production of Epitopes
[0163] Peptides having an amino acid sequence of any of SEQ ID NO: 1, 8, 9, 11-23, 26-29, 32-44, 47-54, 56-63, 66-68 88-253, or 256-588 are synthesized using either FMOC or tBOC solid phase synthesis methodologies. After synthesis, the peptides are cleaved from their supports with either trifluoroacetic acid or hydrogen fluoride, respectively, in the presence of appropriate protective scavengers. After removing the acid by evaporation, the peptides are extracted with ether to remove the scavengers and the crude, precipitated peptide is then lyophilized. Purity of the crude peptides is determined by HPLC, sequence analysis, amino acid analysis, counterion content analysis and other suitable means. If the crude peptides are pure enough (greater than or equal to about 90% pure), they can be used as is. If purification is required to meet drug substance specifications, the peptides are purified using one or a combination of the following: re-precipitation; reverse-phase, ion exchange, size exclusion or hydrophobic interaction chromatography; or counter-current distribution.
[0164] Drug Product Formulation
[0165] GMP-grade peptides are formulated in a parenterally acceptable aqueous, organic, or aqueous-organic buffer or solvent system in which they remain both physically and chemically stable and biologically potent. Generally, buffers or combinations of buffers or combinations of buffers and organic solvents are appropriate. The pH range is typically between 6 and 9. Organic modifiers or other excipients can be added to help solubilize and stabilize the peptides. These include detergents, lipids, co-solvents, antioxidants, chelators and reducing agents. In the case of a lyophilized product, sucrose or mannitol or other lyophilization aids can be added. Peptide solutions are sterilized by membrane filtration into their final container-closure system and either lyophilized for dissolution in the clinic, or stored until use.
[0166] B. Construction of Expression Vectors for Use as Nucleic Acid Vaccines
[0167] The construction of three generic epitope expression vectors is presented below. The particular advantages of these designs are set forth in U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” which has been incorporated by reference in its entirety above.
[0168] A suitable E. coli strain was then transfected with the plasmid and plated out onto a selective medium. Several colonies were grown up in suspension culture and positive clones were identified by restriction mapping. The positive clone was then grown up and aliquotted into storage vials and stored at −70° C.
[0169] A mini-prep (QIAprep Spin Mini-prep: Qiagen, Valencia, Calif.) of the plasmid was then made from a sample of these cells and automated fluorescent dideoxy sequence analysis was used to confirm that the construct had the desired sequence.
[0170] B.1 Construction of pVAX-EP1-IRES-EP2
[0171] Overview:
[0172] The starting plasmid for this construct is pVAX1 purchased from Invitrogen (Carlsbad, Calif.). Epitopes EP 1 and EP2 were synthesized by GIBCO BRL (Rockville, Md.). The IRES was excised from pIRES purchased from Clontech (Palo Alto, Calif.).
[0173] Procedure:
[0174] 1 pIRES was digested with EcoRI and NotI. The digested fragments were separated by agarose gel electrophoresis, and the IRES fragment was purified from the excised band.
[0175] 2 pVAX1 was digested with EcoRI and NotI, and the pVAX1 fragment was gel-purified.
[0176] 3 The purified pVAX1 and IRES fragments were then ligated together.
[0177] 4 Competent E. coli of strain DH5&agr; were transformed with the ligation mixture.
[0178] 5 Minipreps were made from 4 of the resultant colonies.
[0179] 6 Restriction enzyme digestion analysis was performed on the miniprep DNA. One recombinant colony having the IRES insert was used for further insertion of EP1 and EP2. This intermediate construct was called pVAX-IRES.
[0180] 7 Oligonucleotides encoding EP 1 and EP2 were synthesized.
[0181] 8 EP1 was subcloned into pVAX-IRES between AflII and EcoRI sites, to make pVAX-EP1-IRES;
[0182] 9 EP2 was subcloned into pVAX-EP1-IRES between SalI and NotI sites, to make the final construct pVAX-EP1-IRES-EP2.
[0183] 10 The sequence of the EP1-IRES-EP2 insert was confirmed by DNA sequencing.
[0184] B 2. Construction of pVAX-EP1-IRES-EP2-ISS-NIS
[0185] Overview:
[0186] The starting plasmid for this construct was pVAX-EP1-IRES-EP2 (Example 1). The ISS (immunostimulatory sequence) introduced into this construct is AACGTT, and the NIS (standing for nuclear import sequence) used is the SV40 72 bp repeat sequence. ISS-NIS was synthesized by GIBCO BRL. See FIG. 2.
[0187] Procedure:
[0188] 1 pVAX-EP 1-IRES-EP2 was digested with NruI; the linearized plasmid was gel-purified.
[0189] 2 ISS-NIS oligonucleotide was synthesized.
[0190] 3 The purified linearized pVAX-EP1-IRES-EP2 and synthesized ISS-NIS were ligated together.
[0191] 4 Competent E. coli of strain DH5&agr; were transformed with the ligation product.
[0192] 5 Minipreps were made from resultant colonies.
[0193] 6 Restriction enzyme digestions of the minipreps were carried out.
[0194] 7 The plasmid with the insert was sequenced.
[0195] B3. Construction of pVAX-EP2-UB-EP1
[0196] Overview:
[0197] The starting plasmid for this construct was pVAX1 (Invitrogen). EP2 and EP1 were synthesized by GIBCO BRL. Wild type Ubiquitin cDNA encoding the 76 amino acids in the construct was cloned from yeast.
[0198] Procedure:
[0199] 1 RT-PCR was performed using yeast mRNA. Primers were designed to amplify the complete coding sequence of yeast Ubiquitin.
[0200] 2 The RT-PCR products were analyzed using agarose gel electrophoresis. A band with the predicted size was gel-purified.
[0201] 3 The purified DNA band was subcloned into pZERO1 at EcoRV site. The resulting clone was named pZERO-UB.
[0202] 4 Several clones of pZERO-UB were sequenced to confirm the Ubiquitin sequence before further manipulations.
[0203] 5 EP1 and EP2 were synthesized.
[0204] 6 EP2, Ubiquitin and EP1 were ligated and the insert cloned into pVAX1 between BamHI and EcoRI, putting it under control of the CMV promoter.
[0205] 7 The sequence of the insert EP2-UB-EP 1 was confirmed by DNA sequencing.
Example 2[0206] Identification of Useful Epitope Variants
[0207] The 10-mer FLPWHRLFLL (SEQ ID NO. 1) is identified as a useful epitope. Based on this sequence, numerous variants are made. Variants exhibiting activity in HLA binding assays (see Example 3, section 6) are identified as useful, and are subsequently incorporated into vaccines.
[0208] The HLA-A2 binding of length variants of FLPWHRLFLL have been evaluated. Proteasomal digestion analysis indicates that the C-terminus of the 9-mer FLPWHRLFL (SEQ ID NO. 8) is also produced. Additionally the 9-mer LPWHRLFLL (SEQ ID NO. 9) can result from N-terminal trimming of the 10-mer. Both are predicted to bind to the HLA-A*0201 molecule, however of these two 9-mers, FLPWHRLFL displayed more significant binding and is preferred (see FIGS. 3A and B).
[0209] In vitro proteasome digestion and N-terminal pool sequencing indicates that tyrosinase207-216 (SEQ ID NO. 1) is produced more commonly than tyrosinase207-215 (SEQ ID NO. 8), however the latter peptide displays superior immunogenicity, a potential concern in arriving at an optimal vaccine design. FLPWHRLFL, tyrosinase207-215 (SEQ ID NO. 8) was used in an in vitro immunization of HLA-A2+ blood to generate CTL (see CTL Induction Cultures below). Using peptide pulsed T2 cells as targets in a standard chromium release assay it was found that the CTL induced by tyrosinase207-215 (SEQ ID NO. 8) recognize tyrosinase207-216 (SEQ ID NO. 1) targets equally well (see FIG. 3C). These CTL also recognize the HLA-A2+, tyrosinase+ tumor cell lines 624.38 and HTB64, but not 624.28 an HLA-A2− derivative of 624.38 (FIG. 3C). Thus the relative amounts of these two epitopes produced in vivo, does not become a concern in vaccine design.
[0210] CTL Induction Cultures
[0211] PBMCs from normal donors were purified by centrifugation in Ficoll-Hypaque from buffy coats. All cultures were carried out using the autologous plasma (AP) to avoid exposure to potential xenogeneic pathogens and recognition of FBS peptides. To favor the in vitro generation of peptide-specific CTL, we employed autologous dendritic cells (DC) as APCs. DC were generated and CTL were induced with DC and peptide from PBMCs as described (Keogh et al., 2001). Briefly, monocyte-enriched cell fractions were cultured for 5 days with GM-CSF and IL-4 and were cultured for 2 additional days in culture media with 2 g/ml CD40 ligand to induce maturation. 2×106 CD8+-enriched T lymphocytes/well and 2 ×105 peptide-pulsed DC/well were co-cultured in 24-well plates in 2 ml RPMI supplemented with 10% AP, 10 ng/ml IL-7 and 20 IU/ml IL-2. Cultures were restimulated on days 7 and 14 with autologous irradiated peptide-pulsed DC.
[0212] Sequence variants of FLPWHRLFL are constructed as follow. Consistent with the binding coefficient table (see Table 3) from the NIH/BIMAS MHC binding prediction program (see reference in example 3 below), binding can be improved by changing the L at position 9, an anchor position, to V. Binding can also be altered, though generally to a lesser extent, by changes at non-anchor positions. Referring generally to Table 3, binding can be increased by employing residues with relatively larger coefficients. Changes in sequence can also alter immunogenicity independently of their effect on binding to MHC. Thus binding and/or immunogenicity can be improved as follows:
[0213] By substituting F, L, M, W, or Y for P at position 3; these are all bulkier residues that can also improve immunogenicity independent of the effect on binding. The amine and hydroxyl-bearing residues, Q and N; and S and T; respectively, can also provoke a stronger, cross-reactive response.
[0214] By substituting D or E for W at position 4 to improve binding; this addition of a negative charge can also make the epitope more immunogenic, while in some cases reducing cross-reactivity with the natural epitope. Alternatively the conservative substitutions of F or Y can provoke a cross-reactive response.
[0215] By substituting F for H at position 5 to improve binding. H can be viewed as partially charged, thus in some cases the loss of charge can hinder cross-reactivity. Substitution of the fully charged residues R or K at this position can enhance immunogenicity without disrupting charge-dependent cross-reactivity.
[0216] By substituting I, L, M, V, F, W, or Y for R at position 6. The same caveats and alternatives apply here as at position 5.
[0217] By substituting W or F for L at position 7 to improve binding. Substitution of V, I, S, T, Q, or N at this position are not generally predicted to reduce binding affinity by this model (the NIH algorithm), yet can be advantageous as discussed above.
[0218] Y and W, which are equally preferred as the Fs at positions 1 and 8, can provoke a useful cross-reactivity. Finally, while substitutions in the direction of bulkiness are generally favored to improve immunogenicity, the substitution of smaller residues such as A, S, and C, at positions 3-7 can be useful according to the theory that contrast in size, rather than bulkiness per se, is an important factor in immunogenicity. The reactivity of the thiol group in C can introduce other properties as discussed in Chen, J.-L., et al. J. Immunol. 165:948-955, 2000. 5 TABLE 3 9-mer Coefficient Table for HLA-A*0201* HLA Coefficient table for file “A_0201_standard” Amino Acid Type 1st 2nd 3rd 4th 5th 6th 7th 8th 9th A 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 C 1.000 0.470 1.000 1.000 1.000 1.000 1.000 1.000 1.000 D 0.075 0.100 0.400 4.100 1.000 1.000 0.490 1.000 0.003 E 0.075 1.400 0.064 4.100 1.000 1.000 0.490 1.000 0.003 F 4.600 0.050 3.700 1.000 3.800 1.900 5.800 5.500 0.015 G 1.000 0.470 1.000 1.000 1.000 1.000 0.130 1.000 0.015 H 0.034 0.050 1.000 1.000 1.000 1.000 1.000 1.000 0.015 I 1.700 9.900 1.000 1.000 1.000 2.300 1.000 0.410 2.100 K 3.500 0.100 0.035 1.000 1.000 1.000 1.000 1.000 0.003 L 1.700 72.000 3.700 1.000 1.000 2.300 1.000 1.000 4.300 M 1.700 52.000 3.700 1.000 1.000 2.300 1.000 1.000 1.000 N 1.000 0.470 1.000 1.000 1.000 1.000 1.000 1.000 0.015 P 0.022 0.470 1.000 1.000 1.000 1.000 1.000 1.000 0.003 Q 1.000 7.300 1.000 1.000 1.000 1.000 1.000 1.000 0.003 R 1.000 0.010 0.076 1.000 1.000 1.000 0.200 1.000 0.003 S 1.000 0.470 1.000 1.000 1.000 1.000 1.000 1.000 0.015 T 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.500 V 1.700 6.300 1.000 1.000 1.000 2.300 1.000 0.410 14.000 W 4.600 0.010 8.300 1.000 1.000 1.700 7.500 5.500 0.015 Y 4.600 0.010 3.200 1.000 1.000 1.500 1.000 5.500 0.015 *This table and other comparable data that are publicly available are useful in designing epitope variants and in determining whether a particular variant is substantially similar, or is functionally similar.
Example 3[0219] Cluster Analysis (SSX-231-68).
[0220] 1. Epitope Cluster Region Prediction:
[0221] The computer algorithms: SYFPEITHI (internet http://access at syfpeithi.bmi-heidelberg.com/Scripts/MHCServer.dll/EpPredict.htm), based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic; and HLA Peptide Binding Predictions (NIH) (internet http://access at bimas.dcrt.nih.gov/molbio/hla_bin), described in Parker, K. C., et al., J. Immunol. 152:163, 1994; were used to analyze the protein sequence of SSX-2 (GI: 10337583). Epitope clusters (regions with higher than average density of peptide fragments with high predicted MHC affinity) were defined as described fully in U.S. patent application Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,” filed on Apr. 28, 2000. Using a epitope density ratio cutoff of 2, five and two clusters were defined using the SYFPETHI and NIH algorithms, respectively, and peptides score cutoffs of 16 (SYFPETHI) and 5 (NIH) The highest scoring peptide with the NIH algorithm, SSX-241-49, with an estimated halftime of dissociation of >1000 min., does not overlap any other predicted epitope but does cluster with SSX-257-65 in the NIH analysis.
[0222] 2. Peptide Synthesis and Characterization:
[0223] SSX-231-68, YFSKEEWEKMKASEKIFYVYMKRKYEAMTKLGFKATLP (SEQ ID NO. 10) was synthesized by MPS (Multiple Peptide Systems, San Diego, Calif. 92121) using standard solid phase chemistry. According to the provided ‘Certificate of Analysis’, the purity of this peptide was 95%.
[0224] 3. Proteasome Digestion:
[0225] Proteasome was isolated from human red blood cells using the proteasome isolation protocol described in U.S. patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPE DISCOVERY,” filed on Apr. 28, 2000. SDS-PAGE, western-blotting, and ELISA were used as quality control assays. The final concentration of proteasome was 4 mg/ml, which was determined by non-interfering protein assay (Geno Technologies Inc.). Proteasomes were stored at −70° C. in 25 &mgr;l aliquots.
[0226] SSX-231-68 was dissolved in Milli-Q water, and a 2 mM stock solution prepared and 20 &mgr;L aliquots stored at −20° C.
[0227] 1 tube of proteasome (25 &mgr;L) was removed from storage at −70° C. and thawed on ice. It was then mixed thoroughly with 12.5 &mgr;L of 2 mM peptide by repipetting (samples were kept on ice). A 5 &mgr;L sample was immediately removed after mixing and transferred to a tube containing 1.25 &mgr;L 10% TFA (final concentration of TFA was 2%); the T=0 min sample. The proteasome digestion reaction was then started and carried out at 37° C. in a programmable thermal controller. Additional 5 &mgr;L samples were taken out at 15, 30, 60, 120, 180 and 240 min respectively, the reaction was stopped by adding the sample to 1.25 &mgr;L 10% TFA as before. Samples were kept on ice or frozen until being analyzed by MALDI-MS. All samples were saved and stored at −20° C. for HPLC analysis and N-terminal sequencing. Peptide alone (without proteasome) was used as a blank control: 2 &mgr;L peptide +4 &mgr;L Tris buffer (20 mM, pH 7.6)+1.5 &mgr;L TFA.
[0228] 4. MALDI-TOF MS Measurements:
[0229] For each time point 0.3 &mgr;L of matrix solution (10 mg/ml &agr;-cyano4-hydroxycinnamic acid in AcCN/H2O (70:30)) was first applied on a sample slide, and then an equal volume of digested sample was mixed gently with matrix solution on the slide. The slide was allowed to dry at ambient air for 3-5 min. before acquiring the mass spectra. MS was performed on a Lasermat 2000 MALDI-TOF mass spectrometer that was calibrated with peptide/protein standards. To improve the accuracy of measurement, the molecular ion weight (MH+) of the peptide substrate was used as an internal calibration standard. The mass spectrum of the T=120 min. digested sample is shown in FIG. 4.
[0230] 5. MS Data Analysis and Epitope Identification:
[0231] To assign the measured mass peaks, the computer program MS-Product, a tool from the UCSF Mass Spectrometry Facility (http://accessible at prospector.ucsf.edu/ucsfhtml3.4/msprod.htm), was used to generate all possible fragments (N- and C-terminal ions, and internal fragments) and their corresponding molecular weights. Due to the sensitivity of the mass spectrometer, average molecular weight was used. The mass peaks observed over the course of the digestion were identified as summarized in Table 4.
[0232] Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include predicted HLA-A2.1 binding sequences, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 5. 6 TABLE 4 SSX-231-68 Mass Peak Identification. CALCU- LATED MS PEAK MASS (measured) PEPTIDE SEQUENCE (MH+) 988.23 31-37 YFSKEEW 989.08 1377.68 ± 31-40 YFSKEEWEKM 1377.68 2.38 1662.45 ± 31-43 YFSKEEWEKMKAS 1663.90 1.30 2181.72 ± 31-47 YFSKEEWEKMKASEKIF 2181.52 0.85 2346.6 31-48 YFSKEEWEKMKASEICIFY 2344.71 1472.16 ± 38-49 EKMKASEKIFYV 1473.77 1.54 2445.78 ± 31-49* YFSKEEWEKMKASEKIFYV 2443.84 1.18 2607. 31-50 YFSKEEWEKMKASEKIFYVY 2607.02 1563.3 50-61 YMKRKYBAMTKL 1562.93 3989.9 31-61 YFSKEEWEKMKASEKIFYVYMK 3987.77 RKYEAMTKL 1603.74 ± 51-63 MKRKYEAMTKLGF 1603.98 1.53 1766.45 ± 50-63 YMKRKYEAMTKLGF 1767.16 1.53 1866.32 ± 49-63 VYMKRKYEAMTKLGF 1866.29 1.22 4192.6 31-63 YFSKEEWEKMKASEKIFYVYMK 4192.00 RKYEAMTKLGF 4392.1 31-65** YFSKEEWEKMKASEKIFYVYMK 4391.25 RKYEAMTKLGFKA Boldface sequence correspond to peptides predicted to bind to MHC. *On the basis of mass alone this peak could also have been assigned to the peptide 32-50, however proteasomal removal of just the N-terminal amino acid is unlikely. N-terminal sequencing (below) verifies the assignment to 31-49. **On the basis of mass this fragment might also represent 33-68. N-terminal sequencing below is consistent with the assignment to 31-65.
[0233] 7 TABLE 5 Predicted HLA binding by proteasomally generated fragments SEQ ID NO. PEPTIDE HLA SYFPEITHI NIH 11 FSKEEWEKM B*3501 NP† 90 12 KMKASEKIF B*08 17 <5 13 & (14) (K)MKASEKIFY A1 19(19) <5 15 & (16) (M)KASEKIFYV A*0201 22(16) 1017 B*08 17 <5 B*5101 22(13) 60 B*5102 NP 133 B*5103 NP 121 17 & (18) (K)ASEKTFYVY A1 34(19) 14 19 & (20) (K)RKYEAMTKL A*0201 15 <5 A26 15 NP B14 NP 45(60) B*2705 21 15 B*2709 16 NP B*5101 15 <5 21 KYEAMTKLGF A1 16 <5 A24 NP 300 22 YEAMTKLGF B*4403 NP 80 23 EAMTKLGF B*08 22 <5 †No prediction
[0234] As seen in Table 5, N-terminal addition of authentic sequence to epitopes can generate epitopes for the same or different MHC restriction elements. Note in particular the pairing of (K)RKYEAMTKL (SEQ ID NOS 19 and (20)) with HLA-B14, where the 10-mer has a longer predicted halftime of dissociation than the co-C-terminal 9-mer. Also note the case of the 10-mer KYEAMTKLGF (SEQ ID NO. 21) which can be used as a vaccine useful with several MHC types by relying on N-terminal trimming to create the epitopes for HLA-B*4403 and -B*08.
[0235] 6. HLA-A0201 Binding Assay:
[0236] Binding of the candidate epitope KASEKIFYV, SSX-241-49, (SEQ ID NO. 15) to HLA-A2.1 was assayed using a modification of the method of Stauss et al., (Proc Natl Acad Sci USA 89(17):7871-5 (1992)). Specifically, T2 cells, which express empty or unstable MHC molecules on their surface, were washed twice with Iscove's modified Dulbecco's medium (IMDM) and cultured overnight in serum-free AIM-V medium (Life Technologies, Inc., Rockville, Md.) supplemented with human &bgr;2-microglobulin at 3 &mgr;g/ml (Sigma, St. Louis, Mo.) and added peptide, at 800, 400, 200, 100, 50, 25, 12.5, and 6.25 &mgr;g/ml. in a 96-well flat-bottom plate at 3×105 cells/200 &mgr;l/well. Peptide was mixed with the cells by repipeting before distributing to the plate (alternatively peptide can be added to individual wells), and the plate was rocked gently for 2 minutes. Incubation was in a 5% CO2 incubator at 37° C. The next day the unbound peptide was removed by washing twice with serum free RPMI medium and a saturating amount of anti-class I HLA monoclonal antibody, fluorescein isothiocyanate (FITC)-conjugated anti-HLA A2, A28 (One Lambda, Canoga Park, Calif.) was added. After incubation for 30 minutes at 4° C., cells were washed 3 times with PBS supplemented with 0.5% BSA, 0.05% (w/v) sodium azide, pH 7.4-7.6 (staining buffer). (Alternatively W6/32 (Sigma) can be used as the anti-class I HLA monoclonal antibody the cells washed with staining buffer and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat F(ab′) antimouse-IgG (Sigma) for 30 min at 4° C. and washed 3 times as before.) The cells were resuspended in 0.5 ml staining buffer. The analysis of surface HLA-A2.1 molecules stabilized by peptide binding was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, Calif.). If flow cytometry is not to be performed immediately the cells can be fixed by adding a quarter volume of 2% paraformaldehyde and storing in the dark at 4° C.
[0237] The results of the experiment are shown in FIG. 5. SSX-241-49 (SEQ ID NO. 15) was found to bind HLA-A2.1 to a similar extent as the known A2.1 binder FLPSDYFPSV (HBV18-27; SEQ ID NO: 24) used as a positive control. An HLA-B44 binding peptide, AEMGKYSFY (SEQ ID NO: 25), was used as a negative control. The fluoresence obtained from the negative control was similar to the signal obtained when no peptide was used in the assay. Positive and negative control peptides were chosen from Table 18.3.1 in Current Protocols in Immunology p. 18.3.2, John Wiley and Sons, New York, 1998.
[0238] 7. Immunogenicity:
[0239] A. In vivo Immunization of Mice.
[0240] HHD1 transgenic A*0201 mice (Pascolo, S., et al. J. Exp. Med. 185:2043-2051, 1997) were anesthetized and injected subcutaneously at the base of the tail, avoiding lateral tail veins, using 100 &mgr;l containing 100 nmol of SSX-241-49 (SEQ ID NO. 15) and 20 &mgr;g of HTL epitope peptide in PBS emulsified with 50 &mgr;l of IFA (incomplete Freund's adjuvant).
[0241] B. Preparation of Stimulating Cells (LPS Blasts).
[0242] Using spleens from 2 naive mice for each group of immunized mice, un-immunized mice were sacrificed and the carcasses were placed in alcohol. Using sterile instruments, the top dermal layer of skin on the mouse's left side (lower mid-section) was cut through, exposing the peritoneum. The peritoneum was saturated with alcohol, and the spleen was aseptically extracted. The spleen was placed in a petri dish with serum-free media. Splenocytes were isolated by using sterile plungers from 3 ml syringes to mash the spleens. Cells were collected in a 50 ml conical tubes in serum-free media, rinsing dish well. Cells were centrifuged (12000 rpm, 7 min) and washed one time with RPMI. Fresh spleen cells were resuspended to a concentration of 1×106 cells per ml in RPMI-100% FCS (fetal calf serum). 25 g/ml lipopolysaccharide and 7 &mgr;g/ml Dextran Sulfate were added. Cell were incubated for 3 days in T-75 flasks at 37° C., with 5% CO2. Splenic blasts were collected in 50 ml tubes pelleted (12000 rpm, 7 min) and resuspended to 3×107/ml in RPMI. The blasts were pulsed with the priming peptide at 50 &mgr;g/ml, RT 4 hr. mitomycin C-treated at 25 &mgr;g/ml, 37° C., 20 min and washed three times with DMEM.
[0243] C. In vitro Stimulation.
[0244] 3 days after LPS stimulation of the blast cells and the same day as peptide loading, the primed mice were sacrificed (at 14 days post immunization) to remove spleens as above. 3×106 splenocytes were co-cultured with 1×106 LPS blasts/well in 24-well plates at 37° C., with 5% CO2 in DMEM media supplemented with 10% FCS, 5×10−5 M &bgr;-mercaptoethanol, 100 &mgr;g/ml streptomycin and 100 IU/ml penicillin. Cultures were fed 5% (vol/vol) ConA supernatant on day 3 and assayed for cytolytic activity on day 7 in a 51Cr-release assay.
[0245] D. Chromium-Release Assay Measuring CTL Activity.
[0246] To assess peptide specific lysis, 2×106 T2 cells were incubated with 100 &mgr;Ci sodium chromate together with 50 &mgr;g/ml peptide at 37 C for 1 hour. During incubation they were gently shaken every 15 minutes. After labeling and loading, cells were washed three times with 10 ml of DMEM-10% FCS, wiping each tube with a fresh Kimwipe after pouring off the supernatant. Target cells were resuspended in DMEM-10% FBS 1×105/ml. Effector cells were adjusted to 1×107/ml in DMEM-10% FCS and 100 &mgr;l serial 3-fold dilutions of effectors were prepared in U-bottom 96-well plates. 100 &mgr;l of target cells were added per well. In order to determine spontaneous release and maximum release, six additional wells containing 100 &mgr;l of target cells were prepared for each target. Spontaneous release was revealed by incubating the target cells with 100 &mgr;l medium; maximum release was revealed by incubating the target cells with 100 &mgr;l of 2% SDS. Plates were then centrifuged for 5 min at 600 rpm and incubated for 4 hours at 37° C. in 5% CO2 and 80% humidity. After the incubation, plates were then centrifuged for 5 min at 1200 rpm. Supernatants were harvested and counted using a gamma counter. Specific lysis was determined as follows: % specific release=[(experimental release−spontaneous release)/(maximum release—spontaneous release)]×100.
[0247] Results of the chromium release assay demonstrating specific lysis of peptide pulsed target cells are shown in FIG. 6.
[0248] 8. Cross-Reactivity with Other SSX Proteins:
[0249] SSX-241-49 (SEQ ID NO. 15) shares a high degree of sequence identity with the same region of the other SSX proteins. The surrounding regions have also been generally well conserved. Thus the housekeeping proteasome can cleave following V49 in all five sequences. Moreover, SSX41-49 is predicted to bind HLA-A*0201 (see Table 6). CTL generated by immunization with SSX-241-49 cross-react with tumor cells expressing other SSX proteins. 8 TABLE 6 SSX41-49—A*0201 Predicted Binding Family SYFPEITHI NIH SEQ ID NO. Member Sequence Score Score 15 SSX-2 KASEKTFYV 22 1017 26 SSX-1 KYSEKISYV 18 1.7 27 SSX-3 KVSEKIVYV 24 1105 28 SSX-4 KSSEKIVYV 20 82 29 SSX-5 KASEKIIYV 22 175
Example 4[0250] Cluster Analysis (PSMA163-192).
[0251] A peptide, AFSPQGMPEGDLVYVNYARTEDFFKLERDM, PSMA163-192, (SEQ ID NO. 30), containing an A1 epitope cluster from prostate specific membrane antigen, PSMA168-190 (SEQ ID NO. 31) was synthesized using standard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer. After side chain deprotection and cleavage from the resin, peptide first dissolved in formic acid and then diluted into 30% Acetic acid, was run on a reverse-phase preparative HPLC C4 column at following conditions: linear AB gradient (5% B/min) at a flow rate of 4 ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A fraction at time 16.642 min containing the expected peptide, as judged by mass spectrometry, was pooled and lyophilized. The peptide was then subjected to proteasome digestion and mass spectrum analysis essentially as described above. Prominent peaks from the mass spectra are summarized in Table 7. 9 TABLE 7 PSMA163-192 Mass Peak Identification. CALCULATED PEPTIDE SEQUENCE MASS (MU+) 163-177 AFSPQGMPEGDLVYV 1610.0 178-189 NYARTEDFFKLE 1533.68 170-189 PEGDLVYVNYARTEDFFKLE 2406.66 178-191 NYARTEDFFKLERD 1804.95 170-191 PEGDLVYVNYARTEDFFKLERD 2677.93 178-192 NYARTEDFFKLERDM 1936.17 163-176 AFSPQGMPEGDLVY 1511.70 177-192 VNYARTEDFFKLERDM 2035.30 163-179 AFSPQGMPEGDLVYVNY 1888.12 180-192 ARTEDFFKLERDM 1658.89 163-183 AFSPQGMPEGDLVYVNYARTE 2345.61 184-192 DFFKLERDM 1201.40 176-192 YVNYARTEDFFKLERDM 2198.48 167-185 QGMPEGDLVYVNYARTEDF 2205.41 178-186 NYARTEDFF 1163.22 Boldface sequences correspond to peptides predicted to bind to MuG, see Table 8.
[0252] N-Terminal Pool Sequence Analysis
[0253] One aliquot at one hour of the proteasomal digestion (see Example 3 part 3 above) was subjected to N-terminal amino acid sequence analysis by an ABI 473A Protein Sequencer (Applied Biosystems, Foster City, Calif.). Determination of the sites and efficiencies of cleavage was based on consideration of the sequence cycle, the repetitive yield of the protein sequencer, and the relative yields of amino acids unique in the analyzed sequence. That is if the unique (in the analyzed sequence) residue X appears only in the nth cycle a cleavage site exists n−1 residues before it in the N-terminal direction. In addition to helping resolve any ambiguity in the assignment of mass to sequences, these data also provide a more reliable indication of the relative yield of the various fragments than does mass spectrometry.
[0254] For PSMA163-192 (SEQ ID NO. 30) this pool sequencing supports a single major cleavage site after V177 and several minor cleavage sites, particularly one after Y179. Reviewing the results presented in FIGS. 7A-C reveals the following:
[0255] S at the 3rd cycle indicating presence of the N-terminus of the substrate.
[0256] Q at the 5h cycle indicating presence of the N-terminus of the substrate.
[0257] N at the 1st cycle indicating cleavage after V177.
[0258] N at the 3rd cycle indicating cleavage after V175. Note the fragment 176-192 in Table 7.
[0259] T at the 5th cycle indicating cleavage after V177.
[0260] T at the 1st-3rd cycles, indicating increasingly common cleavages after R181, A180 and Y179. Only the last of these correspond to peaks detected by mass spectrometry; 163-179 and 180-192, see Table 7. The absence of the others can indicate that they are on fragments smaller than were examined in the mass spectrum.
[0261] K at the 4th, 8th, and 10th cycles indicating cleavages after E183, Y179, and V177, respectively, all of which correspond to fragments observed by mass spectroscopy. See Table 7.
[0262] A at the 1st and 3rd cycles indicating presence of the N-terminus of the substrate and cleavage after V177, respectively.
[0263] P at the 4th and 8h cycles indicating presence of the N-terminus of the substrate.
[0264] G at the 6th and 10th cycles indicating presence of the N-terminus of the substrate.
[0265] M at the 7th cycle indicating presence of the N-terminus of the substrate and/or cleavage after F185.
[0266] M at the 15th cycle indicating cleavage after V177.
[0267] The 1st cycle can indicate cleavage after D191, see Table 7.
[0268] R at the 4th and 13th cycle indicating cleavage after V177.
[0269] R at the 2nd and 11th cycle indicating cleavage after Y179.
[0270] V at the 2nd, 6th, and 13th cycle indicating cleavage after V175, M169 and presence of the N-terminus of the substrate, respectively. Note fragments beginning at 176 and 170 in Table 7.
[0271] Y at the 1st, 2nd, and 14th cycles indicating cleavage after V175, V177, and presence of the N-terminus of the substrate, respectively.
[0272] L at the 11th and 12th cycles indicating cleavage after V177, and presence of the N-terminus of the substrate, respectively, is the interpretation most consistent with the other data. Comparing to the mass spectrometry results we see that L at the 2nd, 5th, and 9th cycles is consistent with cleavage after F186, E183 or M169, and Y179, respectively. See Table 7.
[0273] Epitope Identification
[0274] Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further analysis. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include a predicted HLA-A1 binding sequence, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 8. 10 TABLE 8 Predicted HLA binding by proteasomally generated fragments SEQ ID NO PEPTIDE HLA SYFPEITHI NIH 32 & (33) (G)MPEGDLVY A*0201 17(27) (2605) V B*0702 20 <5 B*5101 22 314 34 & (35) (Q)GMPEGDLV A1 24(26) <5 Y A3 16(18) 36 B*2705 17 25 36 MPEGDLVY B*5101 15 NP† 37 & (38) (P)EGDLVYVN A1 27(15) 12 Y A26 23(17) NP 39 LVYVNYARTE A3 21 <5 40 & (41) (Y)VNYARTED A26 (20) NP F B*08 15 <5 B*2705 12 50 42 NYARTEDFF A24 NP† 100 Cw*0401 NP 120 43 YARTEDFF B*08 16 <5 44 RTEDFFKLE A1 21 <5 A26 15 NP †No prediction
[0275] HLA-A*0201 Binding Assay:
[0276] HLA-A*0201 binding studies were preformed with PSMA168-177, GMPEGDLVYV, (SEQ ID NO. 33) essentially as described in Example 3 above. As seen in FIG. 8, this epitope exhibits significant binding at even lower concentrations than the positive control peptides. The Melan-A peptide used as a control in this assay (and throughout this disclosure), ELAGIGILTV, is actually a variant of the natural sequence (EAAGIGILTV) and exhibits a high affinity in this assay.
Example 5[0277] Cluster Analysis (PSMA281-310).
[0278] Another peptide, RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG, PSMA281-310, (SEQ ID NO. 45), containing an A1 epitope cluster from prostate specific membrane antigen, PSMA283-307 (SEQ ID NO. 46), was synthesized using standard solid-phase F-moc chemistry on a 433A ABI Peptide synthesizer. After side chain deprotection and cleavage from the resin, peptide in ddH2O was run on a reverse-phase preparative HPLC C18 column at following conditions: linear AB gradient (5% B/min) at a flow rate of 4 ml/min, where eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A fraction at time 17.061 min containing the expected peptide as judged by mass spectrometry, was pooled and lyophilized. The peptide was then subjected to proteasome digestion and mass spectrum analysis essentially as described above. Prominent peaks from the mass spectra are summarized in Table 9. 11 TABLE 9 PSMA281-310 Mass Peak Identification. CALCULATED PEPTIDE SEQUENCE MASS (MH+) 281-297 RGIAEAVGLPSIPVHPI* 1727.07 286-297 AVGLPSIPVHPI** 1200.46 287-297 VGLPSIPVHPI 1129.38 288-297 GLPSIPVHPI† 1030.25 298-310 GYYDAQKLLEKMG‡ 1516.5 298-305 GYYDAQKL§ 958.05 281-305 RGIAEAVGLPSIPVHPIGYYDAQKL 2666.12 281-307 RGIAEAVGLPSIPVHPIGYYDAQKLLE 2908.39 286-307 AVGLPSIPVHPIGYYDAQKLLE¶ 2381.78 287-307 VGLPSTPVHPIGYYDAQKLLE 2310.70 288-307 GLPSIPVHPIGYYDAQKLLE# 2211.57 281-299 RGTAEAVGLPSIPVHPIGY 1947 286-299 AVGLPSIPVHPIGY 1420.69 287-299 VGLPSIPVHPIGY 1349.61 288-299 GLPSIPVHPIGY 1250.48 287-310 VGLPSIPVHPIGYYDAQKLLEKMG 2627.14 288-310 GLPSIPVHPIGYYDAQKLLEKMG 2528.01 Boldface sequences correspond to peptides predicted to bind to MUG, see Table 10. *By mass alone this peak could also have been 296-310 or 288-303. **By mass alone this peak could also have been 298-307. Combination of HPLC and mass spectrometry show that at some later time points this peak is a mixture of both species. †By mass alone this peak could also have been 289-298. By mass alone this peak could also have been 281-295 or 294-306. §By mass alone this peak could also have been 297-303. ¶By mass alone this peak could also have been 285-306. #By mass alone this peak could also have been 288-303. None of these alternate assignments are supported N-terminal pool sequence analysis.
[0279] N-Terminal Pool Sequence Analysis
[0280] One aliquot at one hour of the proteasomal digestion (see Example 3 part 3 above) was subjected to N-terminal amino acid sequence analysis by an ABI 473A Protein Sequencer (Applied Biosystems, Foster City, Calif.). Determination of the sites and efficiencies of cleavage was based on consideration of the sequence cycle, the repetitive yield of the protein sequencer, and the relative yields of amino acids unique in the analyzed sequence. That is if the unique (in the analyzed sequence) residue X appears only in the nth cycle a cleavage site exists n−1 residues before it in the N-terminal direction. In addition to helping resolve any ambiguity in the assignment of mass to sequences, these data also provide a more reliable indication of the relative yield of the various fragments than does mass spectrometry.
[0281] For PSMA281-310 (SEQ ID NO. 45) this pool sequencing supports two major cleavage sites after V287 and I297 among other minor cleavage sites. Reviewing the results presented in FIG. 9 reveals the following:
[0282] S at the 4th and 11th cycles indicating cleavage after V287 and presence of the N-terminus of the substrate, respectively.
[0283] H at the 8th cycle indicating cleavage after V287. The lack of decay in peak height at positions 9 and 10 versus the drop in height present going from 10 to 11 can suggest cleavage after A286 and E285 as well, rather than the peaks representing latency in the sequencing reaction.
[0284] D at the 2nd, 4th, and 7th cycles indicating cleavages after Y299, I297, and V294, respectively. This last cleavage is not observed in any of the fragments in Table 10 or in the alternate assignments in the notes below.
[0285] Q at the 6th cycle indicating cleavage after I297.
[0286] M at the 10th and 12th cycle indicating cleavages after Y299 and I297, respectively.
[0287] Epitope Identification
[0288] Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include a predicted HLA-A1 binding sequence, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 10. 12 TABLE 10 Predicted HLA binding by proteasomally generated fragments: PSMA281-310 SEQ ID NO. PEPTIDE HLA SYFPEITHI NIH 47 & (48) (G)LPSIPVH A*0201 16(24) (24) PI B*0702/B7 23 12 B*5101 24 572 Cw*0401 NP† 20 49 & (50) (P)IGYYDAQ A*0201 (16) <5 KL A26 (20) NP B*2705 16 25 B*2709 15 NP B*5101 21 57 Cw*0301 NP 24 51 & (52) (P)SIPVHPI A1 21(27) <5 GY A26 22 NP A3 16 <5 53 IPVHPIGY B*5101 16 NP 54 YYDAQKLLE A1 22 <5 †No prediction
[0289] As seen in Table 10, N-terminal addition of authentic sequence to epitopes can often generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (G)LPSIPVHPI with HLA-A*0201, where the 10-mer can be used as a vaccine useful with several MHC types by relying on N-terminal trimming to create the epitopes for HLA-B7, -B*5101, and Cw*0401.
[0290] HLA-A*0201 Binding Assay:
[0291] HLA-A*0201 binding studies were preformed with PSMA288-297, GLPSIPVHPI, (SEQ ID NO. 48) essentially as described in Examples 3 and 4 above. As seen in FIG. 8, this epitope exhibits significant binding at even lower concentrations than the positive control peptides.
Example 6[0292] Cluster Analysis (PSMA454-481).
[0293] Another peptide, SSIEGNYTLRVDCTPLMYSLVHLTKEL, PSMA454-481, (SEQ ID NO. 55) containing an epitope cluster from prostate specific membrane antigen, was synthesized by MPS (purity >95%) and subjected to proteasome digestion and mass spectrum analysis as described above. Prominent peaks from the mass spectra are summarized in Table 11. 13 TABLE 11 PSMA454-481 Mass Peak Identification. PEPTIDE SEQUENCE CALCULATED MS PEAK (measured) MASS (MH+) 1238.5 454-464 SSIEGNYTLRV 1239.78 1768.38 ± 0.60 454-469 SSIEGNYTLRVDCTPL 1768.99 1899.8 454-470 SSIEGNYTLRVDCTPLM 1900.19 1097.63 ± 0.91 463-471 RVDCTPLMY 1098.32 2062.87 ± 0.68 454-471* SSIEGNYTLRVDCTPLMY 2063.36 1153 472-481** SLVHNLTKEL 1154.36 1449.93 ± 1.79 470-481 MYSLVHNLTKEL 1448.73 Boldface sequence correspond to peptides predicted to bind to MHC, see Table 12. *On the basis of mass alone this peak could equally well be assigned to the peptide 455-472 however proteasomal removal of just the N-terminal amino acid is considered unlikely. If the issue were important it could be resolved by N-terminal sequencing. **On the basis of mass this fragment might also represent 455-464.
[0294] Epitope Identification
[0295] Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include predicted HLA-A2.1 binding sequences, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 12. 14 TABLE 12 Predicted HLA binding by proteasomally generated fragments SEQ ID NO PEPTIDE HLA SYFPEITHI NIH 56 & (57) (S)IEGNYTLRV A1 (19) <5 A*0201 16(22) <5 58 EGNYTLRV B*5101 15 NP† 59 & (60) (Y)TLRVDCTPL A*0201 20(18) (5) A26 16(18) NP B7 14 40 B8 23 <5 B*2705 12 30 Cw*0301 NP (30) 61 LRVDCTPLM B*2705 20 600 B*2709 20 NP 62 & (63) (L)RVTDCTPLMY A1 32(22) 125 (13.5) A3 25 <5 A26 22 NP B*2702 NP (200) B*2705 13 (NP) (1000) †No prediction
[0296] As seen in Table 12, N-terminal addition of authentic sequence to epitopes can often generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (L)RVDCTPLMY (SEQ ID NOS 62 and (63)) with HLA-B*2702/5, where the 10-mer has substantial predicted halftimes of dissociation and the co-C-terminal 9-mer does not. Also note the case of SIEGNYTLRV (SEQ ID NO 57) a predicted HLA-A*0201 epitope which can be used as a vaccine useful with HLA-B*5101 by relying on N-terminal trimming to create the epitope.
[0297] HLA-A*0201 Binding Assay
[0298] HLA-A*0201 binding studies were preformed, essentially as described in Example 3 above, with PSMA460-469, TLRVDCTPL, (SEQ ID NO. 60). As seen in FIG. 10, this epitope was found to bind HLA-A2.1 to a similar extent as the known A2.1 binder FLPSDYFPSV (HBV18-27; SEQ ID NO: 24) used as a positive control. Additionally, PSMA461-469, (SEQ ID NO. 59) binds nearly as well.
[0299] ELISPOT Analysis: PSMA463-471 (SEQ ID NO. 62)
[0300] The wells of a nitrocellulose-backed microtiter plate were coated with capture antibody by incubating overnight at 4° C. using 50 &mgr;l/well of 4 &mgr;g/ml murine anti-human &ggr;-IFN monoclonal antibody in coating buffer (35 mM sodium bicarbonate, 15 mM sodium carbonate, pH 9.5). Unbound antibody was removed by washing 4 times 5 min. with PBS. Unbound sites on the membrane then were blocked by adding 2001 &mgr;l/well of RPMI medium with 10% serum and incubating 1 hr. at room temperature. Antigen stimulated CD8+ T cells, in 1:3 serial dilutions, were seeded into the wells of the microtiter plate using 100 &mgr;l/well, starting at 2×105 cells/well. (Prior antigen stimulation was essentially as described in Scheibenbogen, C. et al. Int. J. Cancer 71:932-936, 1997. PSMA462-471 (SEQ ID NO. 62) was added to a final concentration of 10 &mgr;g/ml and IL-2 to 100 U/ml and the cells cultured at 37° C. in a 5% CO2, water-saturated atmosphere for 40 hrs. Following this incubation the plates were washed with 6 times 200 &mgr;l/well of PBS containing 0.05% Tween-20 (PBS-Tween). Detection antibody, 50 &mgr;l/well of 2 g/ml biotinylated murine anti-human &ggr;-IFN monoclonal antibody in PBS+10% fetal calf serum, was added and the plate incubated at room temperature for 2 hrs. Unbound detection antibody was removed by washing with 4 times 200 &mgr;l of PBS-Tween. 100 &mgr;l of avidin-conjugated horseradish peroxidase (Pharmingen, San Diego, Calif.) was added to each well and incubated at room temperature for 1 hr. Unbound enzyme was removed by washing with 6 times 200 &mgr;l of PBS-Tween. Substrate was prepared by dissolving a 20 mg tablet of 3-amino 9-ethylcoarbasole in 2.5 ml of N, N-dimethylformamide and adding that solution to 47,5 ml of 0.05 M phosphate-citrate buffer (pH 5.0). 25 &mgr;l of 30% H2O2 was added to the substrate solution immediately before distributing substrate at 100 &mgr;l/well and incubating the plate at room temperature. After color development (generally 15-30 min.), the reaction was stopped by washing the plate with water. The plate was air dried and the spots counted using a stereomicroscope.
[0301] FIG. 11 shows the detection of PSMA463-471 (SEQ ID NO. 62)-reactive HLA-A1+ CD8+ T cells previously generated in cultures of HLA-A1+ CD8+ T cells with autologous dendritic cells plus the peptide. No reactivity is detected from cultures without peptide (data not shown). In this case it can be seen that the peptide reactive T cells are present in the culture at a frequency between 1 in 2.2×104 and 1 in 6.7×104. That this is truly an HLA-A1-restricted response is demonstrated by the ability of anti-HLA-A1 monoclonal antibody to block &ggr;-IFN production; see FIG. 12.
Example 7[0302] Cluster Analysis (PSMA653-687).
[0303] Another peptide, FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY PSMA653-687, (SEQ ID NO. 64) containing an A2 epitope cluster from prostate specific membrane antigen, PSMA660-681 (SEQ ID NO 65), was synthesized by MPS (purity >95%) and subjected to proteasome digestion and mass spectrum analysis as described above. Prominent peaks from the mass spectra are summarized in Table 13. 15 TABLE 13 PSMA653-687 Mass Peak Identification. MS PEAK (measured) PEPTIDE SEQUENCE CALCULATED MASS (MH+) 906.17 ± 0.65 681-687** LPDRPFY 908.05 1287.73 ± 0.76 677-687** DPLGLPDRPFY 1290.47 1400.3 ± 1.79 676-687 IDPLGLPDRPFY 1403.63 1548.0 ± 1.37 675-687 FIDPLGLPDRPFY 1550.80 1619.5 ± 1.51 674-687** AFIDPLGLPDRPFY 1621.88 1775.48 ± 1.32 673-687* RAFIDPLGLPDRPFY 1778.07 2440.2 ± 1.3 653-672 FDKSNPIVLRMMNDQLMFLE 2442.93 1904.63 ± 1.56 672-687* ERAFIDPLGLPDRPFY 1907.19 2310.6 ± 2.5 653-671 FDKSNPIVLRMMNDQLMFL 2313.82 2017.4 ± 1.94 671-687 LERAFIDPLGLPDRPFY 2020.35 2197.43 ± 1.78 653-670 FDKSNPIVLRMMNDQLMF 2200.66 Boldface sequence correspond to peptides predicted to bind to MHC, see Table 13. *On the basis of mass alone this peak could equally well be assigned to a peptide beginning at 654, however proteasomal removal of just the N-terminal amino acid is considered unlikely. If the issue were important it could be resolved by N-terminal sequencing. **On the basis of mass alone these peaks could have been assigned to internal fragments, but given the overall pattern of digestion it was considered unlikely.
[0304] Epitope Identification
[0305] Fragments co-C-terminal with 8-10 amino acid long sequences predicted to bind HLA by the SYFPEITHI or NIH algorithms were chosen for further study. The digestion and prediction steps of the procedure can be usefully practiced in any order. Although the substrate peptide used in proteasomal digest described here was specifically designed to include predicted HLA-A2.1 binding sequences, the actual products of digestion can be checked after the fact for actual or predicted binding to other MHC molecules. Selected results are shown in Table 14. 16 TABLE 14 Predicted lILA binding by proteasomally generated fragments SEQ ID NO PEPTIDE HLA SYFPEITHI NIH 66 & (67) (R)MMNDQLMF A*0201 24 (23) 1360 (722) L A*0205 NP† 71(42) A26 15 NP B*2705 12 50 68 RMMNDQLMF B*2705 17 75 †No prediction
[0306] As seen in Table 14, N-terminal addition of authentic sequence to epitopes can generate still useful, even better epitopes, for the same or different MHC restriction elements. Note for example the pairing of (R)MMNDQLMFL (SEQ ID NOS. 66 and (67)) with HLA-A*02, where the 10-mer retains substantial predicted binding potential.
[0307] HLA-A*0201 Binding Assay
[0308] HLA-A*0201 binding studies were preformed, essentially as described in Example 3 above, with PSMA663-671, (SEQ ID NO. 66) and PSMA662-671, RMMNDQLMFL (SEQ NO. 67). As seen in FIGS. 10, 13 and 14, this epitope exhibits significant binding at even lower concentrations than the positive control peptide (FLPSDYFPSV (HBV18-27); SEQ ID NO: 24). Though not run in parallel, comparison to the controls suggests that PSMA662-671 (which approaches the Melan A peptide in affinity) has the superior binding activity of these two PSMA peptides.
Example 8[0309] Vaccinating with Epitope Vaccines.
[0310] 1. Vaccination with Peptide Vaccines:
[0311] A. Intranodal Delivery
[0312] A formulation containing peptide in aqueous buffer with an antimicrobial agent, an antioxidant, and an immunomodulating cytokine, was injected continuously over several days into the inguinal lymph node using a miniature pumping system developed for insulin delivery (MiniMed; Northridge, CA). This infusion cycle was selected in order to mimic the kinetics of antigen presentation during a natural infection.
[0313] B. Controlled Release
[0314] A peptide formulation is delivered using controlled PLGA microspheres as is known in the art, which alter the pharmacokinetics of the peptide and improve immunogenicity. This formulation is injected or taken orally.
[0315] C. Gene Gun Delivery
[0316] A peptide formulation is prepared wherein the peptide is adhered to gold microparticles as is known in the art. The particles are delivered in a gene gun, being accelerated at high speed so as to penetrate the skin, carrying the particles into dermal tissues that contain pAPCs.
[0317] D. Aerosol Delivery
[0318] A peptide formulation is inhaled as an aerosol as is known in the art, for uptake into appropriate vascular or lymphatic tissue in the lungs.
[0319] 2. Vaccination with Nucleic Acid Vaccines:
[0320] A nucleic acid vaccine is injected into a lymph node using a miniature pumping system, such as the MiniMed insulin pump. A nucleic acid construct formulated in an aqueous buffered solution containing an antimicrobial agent, an antioxidant, and an immunomodulating cytokine, is delivered over a several day infusion cycle in order to mimic the kinetics of antigen presentation during a natural infection.
[0321] Optionally, the nucleic acid construct is delivered using controlled release substances, such as PLGA microspheres or other biodegradable substances. These substances are injected or taken orally. Nucleic acid vaccines are given using oral delivery, priming the immune response through uptake into GALT tissues. Alternatively, the nucleic acid vaccines are delivered using a gene gun, wherein the nucleic acid vaccine is adhered to minute gold particles. Nucleic acid constructs can also be inhaled as an aerosol, for uptake into appropriate vascular or lymphatic tissue in the lungs.
Example 9[0322] Assays for the Effectiveness of Epitope Vaccines.
[0323] 1. Tetramer Analysis:
[0324] Class I tetramer analysis is used to determine T cell frequency in an animal before and after administration of a housekeeping epitope. Clonal expansion of T cells in response to an epitope indicates that the epitope is presented to T cells by pAPCs. The specific T cell frequency is measured against the housekeeping epitope before and after administration of the epitope to an animal, to determine if the epitope is present on pAPCs. An increase in frequency of T cells specific to the epitope after administration indicates that the epitope was presented on pAPC.
[0325] 2. Proliferation Assay:
[0326] Approximately 24 hours after vaccination of an animal with housekeeping epitope, pAPCs are harvested from PBMCs, splenocytes, or lymph node cells, using monoclonal antibodies against specific markers present on pAPCs, fixed to magnetic beads for affinity purification. Crude blood or splenoctye preparation is enriched for pAPCs using this technique. The enriched pAPCs are then used in a proliferation assay against a T cell clone that has been generated and is specific for the housekeeping epitope of interest. The pAPCs are coincubated with the T cell clone and the T cells are monitored for proliferation activity by measuring the incorporation of radiolabeled thymidine by T cells. Proliferation indicates that T cells specific for the housekeeping epitope are being stimulated by that epitope on the pAPCs.
[0327] 3. Chromium Release Assay:
[0328] A human patient, or non-human animal genetically engineered to express human class I MHC, is immunized using a housekeeping epitope. T cells from the immunized subject are used in a standard chromium release assay using human tumor targets or targets engineered to express the same class I MHC. T cell killing of the targets indicates that stimulation of T cells in a patient would be effective at killing a tumor expressing a similar TuAA.
Example 10[0329] Induction of CTL Response with Naked DNA is Efficient by Intra-Lymph Node Immunization.
[0330] In order to quantitatively compare the CD8+ CTL responses induced by different routes of immunization a plasmid DNA vaccine (pEGFPL33A) containing a well-characterized immunodominant CTL epitope from the LCMV-glycoprotein (G) (gp33; amino acids 3341) (Oehen, S., et al. Immunology 99, 163-169 2000) was used, as this system allows a comprehensive assessment of antiviral CTL responses. Groups of 2 C57BL/6 mice were immunized once with titrated doses (200-0.02 &mgr;g) of pEGFPL33A DNA or of control plasmid pEGFP-N3, administered i.m. (intramuscular), i.d. (intradermal), i.spl. (intrasplenic), or i.ln. (intra-lymph node). Positive control mice received 500 pfu LCMV i.v. (intravenous). Ten days after immunization spleen cells were isolated and gp33-specific CTL activity was determined after secondary in vitro restimulation. As shown in FIG. 15, i.m. or i.d. immunization induced weakly detectable CTL responses when high doses of pEFGPL33A DNA (200 &mgr;g) were administered. In contrast, potent gp33-specific CTL responses were elicited by immunization with only 2 &mgr;g pEFGPL33A DNA i.spl. and with as little as 0.2%g pEFGPL33A DNA given i.ln. (FIG. 15; symbols represent individual mice and one of three similar experiments is shown). Immunization with the control pEGFP-N3 DNA did not elicit any detectable gp33-specific CTL responses (data not shown).
Example 11[0331] Intra-Lymph Node DNA Immunization Elicits Anti-Tumor Immunity.
[0332] To examine whether the potent CTL responses elicited following i.ln. immunization were able to confer protection against peripheral tumors, groups of 6 C57BL/6 mice were immunized three times at 6-day intervals with 10 &mgr;g of pEFGPL33A DNA or control pEGFP-N3 DNA. Five days after the last immunization small pieces of solid tumors expressing the gp33 epitope (EL4-33) were transplanted s.c. into both flanks and tumor growth was measured every 3-4d. Although the EL4-33 tumors grew well in mice that had been repetitively immunized with control pEGFP-N3 DNA (FIG. 16), mice which were immunized with pEFGPL33A DNA i.ln. rapidly eradicated the peripheral EL4-33 tumors (FIG. 16).
Example 12[0333] Differences in Lymph Node DNA Content Mirrors Differences in CTL Response Following Intra-Lymph Node and Intramuscular Injection.
[0334] pEFGPL33A DNA was injected i.ln. or i.m. and plasmid content of the injected or draining lymph node was assessed by real time PCR after 6, 12, 24, 48 hours, and 4 and 30 days. At 6, 12, and 24 hours the plasmid DNA content of the injected lymph nodes was approximately three orders of magnitude greater than that of the draining lymph nodes following i.m. injection. No plasmid DNA was detectable in the draining lymph node at subsequent time points (FIG. 17). This is consonant with the three orders of magnitude greater dose needed using i.m. as compared to i.ln. injections to achieve a similar levels of CTL activity. CD8−/− knockout mice, which do not develop a CTL response to this epitope, were also injected i.ln. showing clearance of DNA from the lymph node is not due to CD8+ CTL killing of cells in the lymph node. This observation also supports the conclusion that i.ln. administration will not provoke immunopathological damage to the lymph node.
Example 13[0335] Administration of a DNA Plasmid Formulation of a Therapeutic Vaccine for Melanoma to Humans.
[0336] SYNCHROTOPE TA2M, a melanoma vaccine, encoding the HLA-A2-restricted tyrosinase epitope SEQ ID NO. 1 and epitope cluster SEQ ID NO. 69, was formulated in 1% Benzyl alcohol, 1% ethyl alcohol, 0.5 mM EDTA, citrate-phosphate, pH 7.6. Aliquots of 80, 160, and 320 &mgr;g DNA/ml were prepared for loading into MINIMED 407C infusion pumps. The catheter of a SILHOUETTE infusion set was placed into an inguinal lymph node visualized by ultrasound imaging. The assembly of pump and infusion set was originally designed for the delivery of insulin to diabetics and the usual 17 mm catheter was substituted with a 31 mm catheter for this application. The infusion set was kept patent for 4 days (approximately 96 hours) with an infusion rate of about 25 &mgr;l/hour resulting in a total infused volume of approximately 2.4 ml. Thus the total administered dose per infusion was approximately 200, and 400 &mgr;g; and can be 800 &mgr;g, respectively, for the three concentrations described above. Following an infusion subjects were given a 10 day rest period before starting a subsequent infusion. Given the continued residency of plasmid DNA in the lymph node after administration (as in example 12) and the usual kinetics of CTL response following disappearance of antigen, this schedule will be sufficient to maintain the immunologic CTL response.
Example 14[0337] Additional Epitopes.
[0338] The methodologies described above, and in particular in examples 3-7, have been applied to additional synthetic peptide substrates, leading to the identification of further epitopes as set for the in tables 15-36 below. The substrates used here were designed to identify products of housekeeping proteasomal processing that give rise to HLA-A*0201 binding epitopes, but additional MHC-binding reactivities can be predicted, as discussed above. Many such reactivities are disclosed, however, these listings are meant to be exemplary, not exhaustive or limiting. As also discussed above, individual components of the analyses can be used in varying combinations and orders. The digests of the NY-ESO-1 substrates 136-163 and 150-177 (SEQ ID NOS. 254 and 255, respectively) yielded fragments that did not fly well in MALDI-TOF mass spectrometry. However, they were quite amenable to N-terminal peptide pool sequencing, thereby allowing identification of cleavage sites. Not all of the substrates necessarily meet the formal definition of an epitope cluster as referenced in example 3. Some clusters are so large, e.g. NY-ESO-186-171, that it was more convenient to use substrates spanning only a portion of this cluster. In other cases, substrates were extended beyond clusters meeting the formal definition to include neighboring predicted epitopes. In some instances, actual binding activity may have dictated what substrate was made, as with for example the MAGE epitopes reported here, where HLA binding activity was determined for a selection of peptides with predicted affinity, before synthetic substrates were designed. 17 TABLE 15 GP100: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Comments 609-644 630-638* LPHSSSHWL 88 20/80 16/<5 *The digestion of 609-644 and 622-650 629-638* QLPHSSSHWL 89 21/117 have generated the same epitopes. 614-622 LIYRRRLMK 90 32/20 613-622 SLIYRRRLMK 91 14/<5 29/60 615-622 IYRRRLMK 92 15/<5 622-650 630-638* LPHSSSHWL 93 20/80 16/<5 629-638* QLPHSSSHWL 94 21/117 †Scores are given from the two binding prediction programs referenced aboe (see example 3)
[0339] 18 TABLE 16A MAGE-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digesti SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 86-109 95-102 ESLFRAVI 95 16/<5 93-102 ILESLFRAVI 96 21/<5 20/<5 93-101 ILESLFRAV 97 23/<5 92-101 CILESLFRAV 98 23/55 92-100 CILESLFRA 99 20/138 263-292 263-271 EFLWGPRAL 100 A26 (R 21), A24 (NIH 30) 264-271 FLWGPRAL 101 17/<5 264-273 FLWGPRALAE 102 16/<5 19/<5 265-274 LWGPRALAET 103 16/<5 268-276 PRALAETSY 104 15/<5 267-276 GPRALAETSY 105 15/<5 <15/<5 B4403 (NIH 7); B3501 (NIH 120) 269-277 RALAETSYV 106 18/20 271-279 LAETSYVKV 107 19/<5 270-279 ALAETSYVKV 108 30/427 19/<5<5 272-280 AETSYVKVL 109 15/<5 B.b4403 (NIH 36) 271-280 LAETSYVKVL 110 18/<5 <15/<5 274-282 KVLEYVIKV 111 26/<5 B4403 (NIH 14) 273-282 ETSYVKVLEY 112 28/6 A26 (R 31), B4403 (NIH 14) 278-286 KVLEYVIKV 113 26/743 16/<5
[0340] 19 TABLE 16B MAGE-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 168-193 168-177 SYVLVTCLGL 114 A24 (NIH 300) 169-177 YVLVTCLGL 115 20/32 15/<5 <15/20 170-177 VLVTCLGL 116 17/<5 229-258 240-248 TQDLVQEKY 117 29/<5 239-248 LTQDLVQEKY 118 23/<5 A26 (R 22) 232-240 YGEPRKLLT 119 24/11 243-251 LVQEKYLEY 120 21/<5 21/<5 A26 (R 28) 242-251 DLVQEKYLEY 121 22/<5 19/<5 A26 (R 30) 230-238 SAYGEPRKL 122 21/<5 B5101 (25/121) 272-297 278-286 KVLEYVTKV 123 26/743 16/<5 277-286 VKVLEYVIIKV 124 17/<5 276-284 YVKVLEYVI 125 15/<5 15/<5 17/<5 274-282 TSYVKVLEY 126 26/<5 273-282 ETSYVKVLEY 127 28/6 283-291 VIKVSARVR 128 20/<5 282-291 YVIKVSARVR 129 24/<5 †Scores are given from the two binding prediction programs referenced above (see example 3). R indicates a SYFPEITHI score.
[0341] 20 TABLE 17A MAGE-2: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 107-126 115-122 ELVHFLLL 130 18/<5 113-122 MVELVLIFLLL 131 21/<5 A26 (R 22) 109-116 ISRKMVEL 132 17/<5 108-116 AISRKMVEL 133 25/7 19/<5 16/12 26/<5 107-116 AAISRKMVEL 134 221<5 112-120 KMVELVHFL 135 27/2800 109-117 ISRKMVELV 136 16/<5 108-117 AISRKMVELV 137 24/11 116-124 LVHFLLLKY 138 23/<5 19/<5 A26 (R 26) 115-124 ELVHFLLLKY 139 24/<5 19/5 A26 (R 29) 111-119 RKMVELVHF 140 145-175 158-166 LQLVFGLEV 141 17/168 157-166 YLQLVFGIEV 142 24/1215 159-167 QLVFGIEVV 143 25/32 18/<5 158-167 LQLVFGTEVV 144 18/20 164-172 IEVVEVVPI 145 161<5 163-172 GIEVVEVVPI 146 22/<5 162-170 FGIEVVEVV 147 19/<5 B5101(24/69.212) 154-162 ASEYLQLVF 148 22/68 153-162 KASEYLQLVF 149 15/<5 †Scores are given from the two binding prediction programs referenced above (see example 3). R indicates a SYFPEITHI score.
[0342] 21 TABLE 17B MAGE-2: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence A*0201 A1 A3 B7 B8 Other 213-233 218-225 EEIWEEL 150 22/<5 216-225 APEEKIWEEL 151 15/<5 22/72 216-223 APEEKIWE 152 18/<5 220-228 KIWEELSML 153 26/804 16/<5 16/<5 A26 (R 26) 219-228 EKIWEELSML 154 A26 (R 22) 271-291 271-278 FLWGPRAL 155 17/<5 271-279 FLWGPRALI 156 25/398 16/7 278-286 LIETSYVKV 157 23/<5 277-286 ALIETSYVKV 158 30/427 21/<5 276-284 RALIETSYV 159 18/19 B5101 (20/55) 279-287 IETSYVKVL 160 15/<5 278-287 LLETSYVKVL 161 22/<5 A26 (R 22) †Scores are given from the two binding prediction programs referenced above (see example 3). R indicates a SYFPEITHI score
[0343] 22 TABLE 18 MAGE-3: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 267-286 271-278 FLWGPRAL 162 17/<5 270-278 EFLWGPRAL 163 A26 (R 21); A24 (NIH 30) 271-279 FLWGPRALV 164 27/2655 16/21 5 276-284 RALVETSYV 165 18/19 B5101 (20/55) 272-280 LWGPRALVE 166 15/<5 271-280 FLWGPRALVE 167 15/<5 22/<5 272-281 LWGPRALVET 168 16/<5 †Scores are given from the two binding prediction programs referenced above (see example 3). R indicates a SYFPEITHI score.
[0344] 23 TABLE 19A NY-ESO-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 81-113 82-90 GPESRLLEF 169 16/11 18/<5 22/<5 83-91 PESRLLEFY 170 15/<5 B4403 (NIH 18) 82-91 GPESRLLEFY 171 25/11 84-92 ESRLLEFYL 172 19/8 86-94 RLLEFYLAM 173 21/430 21/<5 88-96 LEFYLAMPF 174 B4403 (NIH 60) 87-96 LLEFYLAMPF 175 <15/45 18/<5 93-102 AMPFATPMEA 176 15/<5 94-102 MPFATPMEA 177 17/<5 101-133 115-123 PLPVPGVLL 178 20/<5 17/<5 16/<5 18/<5 114-123 PPLPVPGVLL 179 23/12 116-123* LPVPGVLL 180 16/<5 Comment 103-112 ELARRSLAQD 181 15/<5 20/<5 *Evidence of the 118-126* VPGVLLKEF 182 17/<5 16/<5 same epitope 117-126* PVPGVLLKEF 183 16/<5 obtained from two 116-145 116-123* LPVPGVLL 184 16/<5 digests. 127-135 TVSGNILTI 185 21/<5 19/<5 126-135 FTVSQNILTI 186 20/<5 120-128 GVLLKEFTV 187 20/130 18/<5 121-130 VLLKEFTVSG 188 17/<5 18/<5 122-130 LLKEFTVSG 189 20/<5 18/<5 118-126* VPGVLLKEF 190 17/<5 16/<5 117-126* PVPGVLLKEF 191 16/<5 †Scores are given from the two binding prediction programs referenced above (see example 3).
[0345] 24 TABLE 19B NY-ESO-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 136-163 139-147 AADHRQLQL 192 17/<5 17/<5 22/<5 (SEQ ID NO 148-156 SISSCLQQL 193 24/7 A26 (R 25) 254) 147-156 LSISSCLQQL 194 18/<5 138-147 TAADHRQLQL 195 18/<5 150-177 161-169 WITQCFLPV 196 18/84 (SEQ ID NO 157-165 SLLMWITQC 197 18/42 17/<5 255) 150-158 SSCLQQLSL 198 15/<5 154-162 QQLSLLMWI 199 15/50 151-159 SCLQQLSLL 200 18/<5 150-159 SSCLQQLSLL 201 16/<5 163-171 TQCFLPVFL 202 <15/12 162-171 ITQCFLPVFL 203 18/<5 A26 (R 19) †Scores are given from the two binding prediction programs referenced above (see example 3). R indicates SYFPEITHI score
[0346] 25 TABLE 20 PRAME: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 211-245 219-227 PMQDIKMIL 204 16/<5 16/n.d. A26 (R 20) 218-227 MPMQDIKMIL 205 <15/240 411-446 428-436 QHLIGLSNL 206 18/<5 427-436 LQHLIGLSNL 207 16/8 429-436 HLIGLSNL 208 17/<5 B15 (R 21) 431-439 IGLSNLTHV 209 18/7 B*5101 (R 22) 430-439 LIGLSNLTHV 210 24/37 †Scores are given from the two binding prediction programs reference above (see example 3). R indicates a SYFPEITHI score.
[0347] 26 TABLE 21 PSA: Preferred Epitopes Revealed by Housekeeping SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 42-77 53-61 VLVHPQWVL 211 22/112 <15/6 17<5 52-61 GVLVHPQWVL 212 17/21 16/<5 <15/30 A26 (R 18) 52-60 GVLVHPQWV 213 17/124 59-67 WVLTAAHCI 214 15/16 54-63 LVHPQWVLTA 215 19/<5 20/<5 A26 (R 16) 53-62 VLVHPQWVLT 216 17/22 54-62 LVHPQWVLT 217 17/n.d. 55-95 66-73 CIRNKSVI 218 26/20 65-73 HCIRNKSVI 219 <15/16 56-64 HPQWVLTAA 220 18/<5 63-72 AAHCIRNKSV 221 17/<5 †Scores are given from the two binding prediction programs referenced above (see example 3). R indicates a SYFPEITHI score.
[0348] 27 TABLE 22 PSCA: Preferred Epitopes Revealed by Housekeeping Proteasoe Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 93-123* 116-123 LLWGPGQL 222 16/<5 115-123 LLLWGPGQL 223 <15/18 114-123 GLLLWGPGQL 224 <15/10 99-107 ALQPAAAIL 225 26/9 22/<5 <15/12 16/<5 A26 (R 19) 98-107 HALQPAAAIL 226 18/<5 <15/12 *L123 is the C-terminus of the natural prote †Scores are given from the two binding prediction programs referenced above (see example 3).
[0349] 28 TABLE 23 Tyrosinase: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 128-157 128-137 APEKDKFFAY 227 29/6 15/<5 B4403 (NIH 14) 129-137 PEKDKFFAY 228 18/<5 21/<5 130-138 EKDKFFAYL 229 15/<5 131-138 KDKIFFAYL 230 20/<5 197-228 205-213 PAFLPWIIRL 231 15/<5 204-213 APAFLPWHRL 232 23/360 207-216 FLPWIIRLFLL 1 25/1310 <15/18 208-216 LPWHRLFLL 9 17/26 20/80 24/16 214-223 FLLRWEQEIQ 233 15/<5 212-220 RLFLLRWEQ 234 16/<5 191-211 191-200 GSETWRDIDF 235 18/68 192-200 SEIWRDIDF 236 16/<5 B4403 (NIH 400) 207-230 207-215 FLWIIRLFL 8 22/540 <15/6 17/<5 466-484 473-481 RTWSWLLGA 237 19/13 15/<5 476-497 476-484 SWLLGAAMV 238 18/<5 477-486 WLLGAAMVGA 239 21/194 18/<5 478-486 LLGAAMVGA 240 19/19 16/<5 †Scores are given from the two binding prediction programs referenced above (see example 3).
[0350] 29 TABLE 24 PSMA: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion SEQ ID HLA Binding Predictions (SYFPEITHI/NIH)† Substrate Epitope Sequence NO A*0201 A1 A3 B7 B8 Other 1-30 4-12 LLHETDSAV 241 25/485 15/<5 13-21 ATARRPRWL 242 18/<5 18/<5 53-80 53-61 TPKHNMKAF 243 24/<5 64-73 ELKAENIKKF 244 17/<5 A26 (R 30) 69-77 NIKKFLH1NF 245 A26 (R 27) 68-77 ENIKKFLH1NF 246 A26 (R 24) 215-244 220-228 AGAKGVILY 247 25/<5 457-489 468-477 PLMYSLVLJNL 248 22/<5 469-477 LMYSLVHNL 249 27/193 <15/9 463-471 RVDCTPLMY 250 32/125 25/<5 A26 (R 22) 465-473 DCTPLMYSL 251 A26 (R 22) 503-533 507-515 SGMPRISKL 252 21/<5 21<5 506-515 FSGMPRISKL 253 17/<5 1This was reported as Y in the SWISSPROT database. †Scores are given from the two binding prediction programs referenced above (see example 3).
[0351] 30 TABLE 25A MAGE-1: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Binding Prediction Substrate Epitope Sequence Seq. Id No. HLA type SYFPEITHI NIH Mage-1 119-146 125-132 KAEMLESV 256 B5101 19 n.a. 124-132 TKAEMLESV 257 A0201 20 <5 123-132 VTKAEMLESV 258 A0201 20 <5 128-136 MLESVIKNY 259 A1 28 45 A26 24 n.a. A3 17 5 127-136 EMLESVIKNY 260 A1 15 <1.0 A26 23 <1.0 125-133 KAEMLESVI 261 B5101 23 100 A24 N.A. 4 Mage-1 143-170 146-153 KASESLQL 262 B08 16 <1.0 B5101 17 N.A. 145-153 GKASESLQL 263 B2705 17 1 B2709 16 N.A. 147-155 ASESLQLVF 264 A1 22 68 153-161 LVFGIDVKE 265 A26 16 N.A. A3 16 <1.0
[0352] 31 TABLE 25B MAGE-1: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Binding Prediction +HL,42 Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH Mage-1 99-125 114-121 LLKYRARE 266 B8 25 <1.0 106-113 VADLVGFL 267 B8 16 <1.0 B5101 21 N.A. 105-113 KVADLVGFL 268 A0201 23 44 A26 25 N.A. A3 16 <5 B0702 14 20 B2705 14 30 107-115 ADLVGFLLL 269 A0201 17 <5 B0702 15 <5 B2705 16 1 106-115 VADLVGFLLL 270 A0201 16 <5 A1 22 3 114-123 LLKYRAREPV 271 A0201 20 2
[0353] 32 TABLE 26 MAGE-3: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Predition Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH Mage-3 267-295 271-278 FLWGPRAL 162 B08 17 <5 270-278 EFLWGPRAL 163 A26 21 N.A. A24 N.A. 30 B1510 16 N.A. 271-279 FLWGPRALV 164 A0201 27 2655 A3 16 2 278-286 LVETSYVKV 272 A0201 19 <1.0 A26 17 N.A. 277-286 ALVETSYVKV 273 A0201 28 428 A26 16 <5 A3 18 <5 285-293 KVLHHMVKI 274 A0201 19 27 A3 19 <5 276-284 RALVETSYV 165 A0201 18 20 283-291 YVKVLHHMV 275 A0201 17 <1.0 275-283 PRALVETSY 276 A1 17 <1.0 274-283 GPRALVETSY 277 A1 15 <1.0 278-287 LVETSYVKVL 278 A0201 18 <1.0 272-281 LWGPRALVET 168 A0201 16 <1.0 271-280 FLWGPRALVE 167 A3 22 <5
[0354] 33 TABLE 27A Fibronectin ED-B: Preferred Epitopes Revealed by Housekeeping Pro- teasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH ED-B 14′-21* 4′-5** TIIPEVPQL\ 279 A0201 27 7 A26 28 N.A. A3 17 <5 B8 15 <5 B1510 15 N.A. B2705 17 10 B2709 15 N.A. 5′-5** DTIIPEVPQL† 280 A0201 20 <5 A26 32 N.A. 1-10 EVPQLTDLSF 281 A26 29 N.A. *This substrate contains the 14 amino acids from fibronectin flanking ED-B to the N-terminal side. **These peptides span the junction between the N-terminus of the ED-B domain and the rest of fibronectin. †The italicized lettering indicates sequence outside the ED-B domain.
[0355] 34 TABLE 27B Fibronectin ED-B: Preferred Epitopoes Revealed by Housekeeping Proteasome Digestion Seq. Binding Prediction Sub- Epi- ID HLA strate tope Sequence No. type SYFPEITHI NIH ED-B 23-30 TPLNSSTI 282 B5101 22 N.A. 8-35 18-25 IGLRWTPL 283 B5101 18 N.A. 17-25 SIGLRWTPL 284 A0201 20 5 A26 18 N.A. B08 25 <5 25-33 LNSSTIIGY 285 A1 19 <5 A26 16 <5 24-33 PLNSSTIIGY 286 A1 20 <5 A26 24 N.A. A3 16 <5 23-31 TPLNSSTII 287 B0702 17 8 B5101 25 440
[0356] 35 TABLE 27C Fibronectin ED-B: Preferred Epitopes Revealed by Housekeeping Pro- teasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH ED-B 20-49 31-38 IGYRITVV 288 B5101 25 N.A. 30-38 IIGYRITVV 289 A0201 23 15 A3 17 <1.0 B08 15 <1.0 B5101 15 <1.0 29-38 TIIGYRITVV 290 A0201 26 9 A26 18 N.A. A3 18 N.A. 23-30 TPLNSSTI 282 B5101 22 N.A. 25-33 LNSSTIIGY 285 A1 19 <5 A26 16 N.A. 24-33 PLNSSTIIGY 286 A26 24 N.A. A3 16 <5 31-39 IGYRITVVA 291 A3 17 <5 30-39 IIGYRITVVA 292 A0201 15 <5 A3 18 <5 23-31 TPLNSSTII 287 B0702 17 8 B5101 25 440
[0357] 36 TABLE 28A CEA: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH CEA 176-202 184-191 SLPVSPRL 293 B08 19 <5 183-191 QSLPVSPRL 294 A0201 15 <5 B1510 15 B2705 18 10 B2709 15 186-193 PVSPRLQL 295 B08 18 <5 185-193 LPVSPRLQL 296 B0702 26 180 B08 16 <5 B5101 19 130 184-193 SLPVSPRLQL 297 A0201 23 21 A26 18 N.A. A3 18 <5 185-192 LPVSPRLQ 298 B5101 17 N.A. 192-200 QLSNGNRTL 299 A0201 21 4 A26 16 N.A. A3 19 <5 B08 17 <5 B1510 15 191-200 LQLSNGNRTL 300 A0201 16 3 179-187 WVNNQSLPV 301 A0201 16 28 186-194 PVSPRLQLS 302 A26 17 N.A. A26 A3 15 <5
[0358] 37 TABLE 28B CEA: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH CEA 354-380 362-369 SLPVSPRL 303 B08 19 <1.0 361-369 QSLPVSPRL 304 A0201 15 <1.0 B2705 18 10 B2709 15 364-371 PVSPRLQL 305 B08 18 <1.0 363-371 LPVSPRLQL 306 B0702 26 180 B08 16 <1.0 B5101 19 130 362-371 SLPVSPRLQL 307 A0201 23 21 A26 16 N.A. A24 N.A. 6 A3 18 <5 363-370 LPVSPRLQ 308 B5101 17 N.A. 370-378 QLSNDNRTL 309 A0201 22 4 A26 16 N.A. A3 17 <1.0 B08 17 <1.0 369-378 LQLSNDNRTL 310 A0201 16 3 357-365 WVNNQSLPV 311 A0201 16 28 360-368 NQSLPVSPR 312 B2705 14 100
[0359] 38 TABLE 28C CEA: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Seq. Binding Prediction Substrate Epitope Sequence ID No. HLA type SYFPEITHI NIH CEA 532-558 540-547 SLPVSPRL 313 B08 19 <5 539-547 QSLPVSPRL 314 A0201 15 <5 B1510 15 <5 B2705 18 10 B2709 15 542-549 PVSPRLQL 315 B08 18 <5 541-549 LPVSPRLQL 316 B0702 26 180 B08 16 <1.0 B5101 19 130 540-549 SLPVSPRLQL 317 A0201 23 21 A26 18 N.A. A3 18 <5 541-548 LPVSPRLQ 318 B5101 17 N.A. 548-556 QLSNGNRTL 319 A0201 24 4 A26 16 N.A. A3 19 <1.0 B08 17 <1.0 B1510 15 547-556 LQLSNGNRTL 320 A0201 16 3 535-543 WVNGQSLPV 321 A0201 18 28 A3 15 <1.0 533-541 LWWVNGQSL 322 A0201 15 <5
[0360] 39 TABLE 28D CEA: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Seq. ID Binding Prediction Substrate Epitope Sequence No. HLA type SYFPEITHI NIH CEA 532-558 532-541 YLWWVNGQSL 323 A0201 25 816 (continued) A26 18 N.A. 538-546 GQSLPVSPR 324 B2705 17 100
[0361] 40 TABLE 29A HER2/NEU: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH Her-2 25-52 30-37 DMKLRLPA 325 B08 19 8 28-37 GTDMKLRLPA 326 A1 23 6 42-49 HLDMLRHL 327 B08 17 <5 41-49 THLDMLRHL 328 A0201 17 <5 B1510 24 N.A. 40-49 ETHLDMLRHL 329 A26 29 N.A. 36-43 PASPETHL 330 B5101 17 N.A. 35-43 LPASPETHL 331 A0201 15 <5 B5101 20 130 B5102 N.A. 100 34-43 RLPASPETHL 332 A0201 20 21 38-46 SPETHLDML 333 A0201 15 <5 B0702 20 24 B08 18 <5 B5101 18 <5 37-46 ASPETHLDML 334 A0201 18 <5 42-50 HLDMLRHLY 335 A1 29 25 A26 20 N.A. A3 17 4 41-50 THLDMLRHLY 336 A1 18 <1.0
[0362] 41 TABLE 29B HER2/NEU: Preferred Epitopes Revealed by Housekeeing Proteasome Di- gestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH Her-2 705-732 719-726 ELRKVKVL 337 B08 24 16 718-726 TELRKVKVL 338 A0201 16 1 B08 22 <5 B5101 16 <5 717-726 ETELRKVKVL 339 A1 18 2 A26 28 6 715-723 LKETELRKV 340 A0201 17 <5 B5101 15 <5 714-723 ILKETELRKV 341 A0201 29 8 712-720 MRILKETEL 342 A0201 15 <5 B08 22 <5 B2705 27 2000 B2709 21 N.A. 711-720 QMRILKETEL 343 A1 18 5 B0702 13 40 717-725 ETELRKVKV 344 A1 18 5 A26 18 N.A. 716-725 KETELRKVKV 345 A0201 16 19 706-714 MPNQAQMRI 346 B0702 16 8 B5101 22 629 705-714 AMPNQAQMRI 347 A0201 18 8 706-715 MPNQAQMRIL 348 B0702 20 80
[0363] 42 TABLE 29C HER2/NEU: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH Her-2 954-982 966-973 RPRFRELV 349 B08 20 24 B5101 18 N.A. 965-973 CRPRFRELV 350 B2709 18 968-976 RFRELVSEF 351 A26 25 N.A. A24 N.A. 32 A3 15 <5 B08 16 <5 B2705 19 967-976 PRERELVSEF 352 A26 18 N.A. 964-972 ECRPRFREL 353 A26 21 N.A. A24 N.A. 6 B0702 15 40 B8 27 640 B1510 16 <5
[0364] 43 TABLE 30 NY-ESO-1: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH NY-ESO-1 51-77 67-75 GAASGLNGC 354 A0201 15 <5 52-60 RASGPGGGA 355 B0702 15 <5 64-72 PHGGAASGL 356 B1510 21 N.A. 63-72 GPHGGAASGL 357 B0702 22 80 60-69 APRGPHGGAA 358 B0702 23 60
[0365] 44 TABLE 31A PRAME: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PRAME 103-135 112-119 VRPRRWKL 359 B08 19 111-110 EVRPRRWKL 360 A26 27 N.A. A24 N.A. 5 A3 19 N.A. B0702 15 (B7)300.00 B08 26 160 113-121 RPRRWKLQV 361 B0702 21 (B7)40.00 B5101 19 110 114-112 PRRWKLQVL 362 B08 N.A. 160 B2705 23 200 113-122 RPRRWKLQVL 363 B0702 24 (B7)800.00 B8 N.A. 160 B5101 N.A. 61 B5102 N.A. 61 A24 N.A. 10 116-124 RWKLQVLDL 364 B08 22 <5 B2705 17 3 115-124 RRWKLQVLDL 365 A0201 16 <5 PRAME 161-187 174-182 PVEVLVDLF 366 A26 25 N.A.
[0366] 45 TABLE 31B PRAME: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PRAME 185-215 199-206 VKRKKNVL 367 B08 27 8 198-206 KVKRKKNVL 368 A0201 16 <1.0 A26 20 N.A. A3 22 <1.0 B08 30 40 B2705 16 197-206 EKVKRKKNVL 369 A26 15 N.A. 198-205 KVKRKKNV 370 B08 20 6 201-208 RKKNVLRL 371 B08 20 <5 200-208 KRKKNVLRL 372 A0201 15 <1.0 A26 15 N.A. B0702 15 <1.0 B08 21 <1.0 B2705 28 B2709 25 199-208 VKRKKNVLRL 373 A0201 16 <1.0 B0702 16 4 189-196 DELFSYLI 374 B5101 15 N.A. 205-213 VLRLCCKKL 375 A0201 22 3 A26 17 N.A. B08 25 8 204-213 NVLRLCCKKL 376 A0201 17 7 A26 19 N.A.
[0367] 46 TABLE 31C PRAME: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PRAME 185-215 194-202 YLIEKVKRK 377 A0201 20 <1.0 (continued) A26 18 N.A. A3 25 ta 68 B08 20 <1.0 B2705 17 PRAME 71-98 74-81 QAWPFTCL 378 B5101 17 n.a. 73-81 VQAWPFTCL 379 A0201 14 7 A24 n.a. 5 B0702 13 30 81-88 LPLGVLMK 381 B5101 18 n.a. 80-88 CLPLGVLMK 382 A0201 17 <1.0 A3 27 120 79-88 TCLPLGVLMK 383 A1 12 10 A3 19 3 84-92 GVLMKGQHL 384 A0201 18 7 A26 21 n.a. B08 21 4 81-89 LPLGVLMKG 385 B5101 20 2 80-89 CLPLGVLMKG 386 A0201 16 <1.0 76-85 WPFTCLPLGV 387 B0702 18 4
[0368] 47 TABLE 31D PRAME: Preferred Epitopes Revealed by Housekeeping Proteasome Di- gestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PRAME 39-65 51-59 ELFPPLFMA 388 A0201 19 18 A26 23 N.A. 49-57 PRELFPPLF 389 B2705 22 B2709 19 48-57 LPRELFPPLF 390 B0702 19 4 50-58 RELFPPLFM 391 B2705 16 B2705 15 49-58 PRELFPPLFM 392 A1 16 <1.0
[0369] 48 TABLE 32 PSA: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PSA 232-258 239-246 RPSLYTKV 393 B5101 21 N.A. 238-246 ERPSLYTKV 394 B2705 15 60 236-243 LPERPSLY 395 B5101 18 N.A. 235-243 ALPERPSLY 396 A1 19 <1.0 A26 22 N.A. A3 26 6 B08 16 <1.0 B2705 11 15 B2709 19 N.A. 241-249 SLYTKVVHY 397 A0201 20 <1.0 A1 19 <1.0 A26 25 N.A. A3 26 60 B08 20 <1.0 B2705 13 75 240-249 PSLYTKVVHY 398 A1 20 <1.0 A26 16 N.A. 239-247 RPSLYTKVV 399 B0702 21 4 B5101 23 110
[0370] 49 TABLE 33A PSMA: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PSMA 202-228 211-218 GNKVKNAQ 400 B08 22 <5 202-209 IARYGKVF 401 B08 18 <5 217-225 AQLAGAKGV 402 A0201 16 26 207-215 KVFRGNKVK 403 A3 32 15 211-219 GNKVKNAQL 404 B8 33 80 B2705 17 20 PSMA 255-282 269-277 TPGYPANEY 405 A1 16 <5 268-277 LTPGYPANEY 406 A1 21 1 A26 24 N.A. 271-279 GYPANEYAY 407 A1 15 <5 270-279 PGYPANEYAY 408 A1 19 <5 266-274 DPLTPGYPA 409 B0702 21 3 B5101 17 20 PSMA 483-509 492-500 SLYESWTKK 410 A0201 17 <5 A3 27 150 B2705 18 150 491-500 KSLYESWTKIK 411 A3 16 <5 486-494 EGFEGKSLY 412 A1 19 <5 A26 21 N.A. B2705 16 <5 485-494 DEGFEGKSLY 413 A1 17 <5 A26 17 N.A. 498-506 TKKSPSPEF 414 B08 17 <5
[0371] 50 TABLE 33B PSMA: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PSMA 483-509 497-506 WTKKSPSPEF 415 A26 24 N.A. (continued) 492-501 SLYESWTKKS 416 A0201 16 <5 A3 16 <5 PSMA 721-749 725-732 WGEVKRQI 417 B08 17 <5 B5101 17 N.A. 724-732 AWGEVKRQI 418 B5101 15 6 723-732 KAWGEVKRQI 419 A0201 16 <1.0 723-730 KAWGEVKR 420 B5101 15 N.A. 722-730 SKAWGEVKR 421 B2705 15 <5 731-739 QIYVAAFTV 422 A0201 21 177 A3 21 <1.0 B5101 15 5 733-741 YVAAFTVQA 423 A0201 17 6 A3 20 <1.0 725-733 WGEVKRQIY 424 A1 26 11 727-735 EVKRQLYVA 425 A26 22 N.A. A3 18 <1.0 738-746 TVQAAAETL 426 A26 18 N.A. A3 19 <1.0 737-746 FTVQAAAETL 427 A0201 17 <1.0 A26 19 N.A.
[0372] 51 TABLE 33C PSMA: Preferred Epitopes Revealed by Housekeeping Proteasome Diges- tion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH PSMA 721-749 729-737 KRQIYVAAF 428 A26 16 N.A. (continued) B2705 24 3000 B2709 21 N.A. 721-729 PSKAWGEVK 429 A3 20 <1.0 723-731 KAWGEVKRQ 430 B5101 16 <1.0 PSMA 95-122 100-108 WKEFGLDSV 431 A0201 16 <5 99-108 QWKEFGLDSV 432 A0201 17 <5 102-111 EFGLDSVELA 433 A26 16 N.A.
[0373] 52 TABLE 34A SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 117-143 126-134 ELRQKESKL 434 A0201 20 <5 A26 26 N.A. A3 17 <5 B07f02 13 (B7)40.00 B8 34 320 125-134 AELRQKESKL 435 A0201 16 <5 133-141 KLQENRKII 436 A0201 20 61 SCP-1 281-308 298-305 QLEEKTKL 437 B08 28 2 297-305 NQLEEKTKL 438 A0201 15 33 B2705 19 200 288-296 LLEESRDKV 439 A0201 25 15 B2705 19 200 287-296 FLLEESRDKV 440 A0201 27 2378 291-299 ESRDKVNQL 441 A26 17 N.A. B08 29 240 290-299 EESRDKVNQL 442 A26 17 N.A. SCP-1 471-498 475-483 EKEVHDLEY 443 A1 31 11 A26 17 N.A. 474-483 REKEVHDLEY 444 A1 21 <1.0 480-488 DLEYSYCHY 445 A1 26 45 A26 30 N.A. A3 16 <5 477-485 EVHDLEYSY 446 A1 15 1
[0374] 53 TABLE 34B SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Eptiope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 471-498 477-485 EVHDLEYSY A26 29 N.A. (continued) A3 19 <1.0 477-486 EVHDLEYSYC 447 A26 22 N.A. SCP-1 493-520 502-509 KLSSKREL 448 B08 26 4 508-515 ELKNTEYF 449 B08 24 <1.0 507-515 RELKNTEYF 450 B2705 18 45 B4403 N.A. 120 496-503 KRGQRPKL 451 B08 18 <1.0 494-503 LPKRGQRPKL 452 B0702 22 120 B8 N.A. 16 B5101 N.A. 130 B3501 N.A. 60 509-517 LKNTEYFTL 453 A0201 15 <5 508-517 ELKNTEYFTL 454 A0201 18 <1.0 A26 27 N.A. A3 16 <1.0 506-514 KRELKNTEY 455 A1 26 2 B2705 26 3000 502-510 KLSSKRELK 456 A3 25 60 498-506 GQRPKLSSK 457 A3 22 4 B2705 18 200 497-506 RGQRPKLSSK 458 A3 22 <1.0 500-508 RPKLSSKRE 459 B08 18 <1.0
[0375] 54 TABLE 34C SCP-1: Preferred Epitope Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 570-596 573-580 LEYVREEL 560 B08 19 <5 572-580 ELEYVREEL 461 A0201 17 <1.0 A26 23 N.A. A24 N.A. 9 B08 20 N.A. 571-580 N ELEYVREEL 462 A0201 16 4 579-587 ELKQKRDEV 463 A0201 19 <1.0 A26 18 N.A. B08 29 48 575-583 YVREELKQK 464 A26 17 N.A. A3 27 2 SCP-1 618-645 632-640 QLNVYEIKV 465 A0201 24 70 630-638 SKQLNVYEI 466 A0201 17 <5 628-636 AESKQLNVY 467 A1 19 <5 A26 16 N.A. 627-636 TAESKQLNVY 468 A1 26 45 A26 15 N.A.
[0376] 55 TABLE 34D SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Eptiope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 633-660 638-645 IKVNKLEL 469 B08 21 <1.0 637-345 EIKVNKLEL 470 A0201 17 <1.0 A26 26 N.A. B08 28 8 B1510 15 N.A. 636-645 YEIKVNKLEL 471 A0201 17 2 642-650 KLELELESA 472 A0201 20 1 A3 16 <1.0 635-643 VYEIKVNKL 473 A0201 18 <1.0 A24 N.A. 396 B08 22 <1.0 634-643 NVYEIKVNKL 474 A0201 24 56 A26 25 N.A. A24 N.A. 6 A3 15 <5 B0702 11 (B7) 20 B08 N.A. 6 646-654 ELESAKQKF 475 A26 27 N.A. SCP-1 640-668 642-650 KLELELESA 476 A0201 20 1 A3 16 <1.0 646-654 ELESAKQKF 477 A26 27 N.A.
[0377] 56 TABLE 34E SCP-1: Preferred Eptiopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Eptiope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 768-796 771-778 KEKLKREA 478 B08 21 <5 777-785 EAKENTATL 479 A0201 18 <5 A26 18 N.A. A24 N.A. 5 B0702 13 12 B08 28 48 B5101 20 121 776-785 REAKENTATL 480 A0201 16 <5 773-782 KLKREAKENT 481 A3 17 <5 SCP-192-125 112-119 EAEKIKKW 482 B5101 17 N.A. 101-109 GLSRVYSKL 483 A0201 23 32 A26 22 N.A. A24 N.A. 6 A3 17 3 B08 17 <1.0 100-109 EGLSRVYSKL 484 A26 21 N.A. A24 N.A. 9 108-116 KLYKEAEKI 485 A0201 22 57 A3 20 9 B5101 18 5 98-106 NSEGLSRVY 486 A1 31 68 97-106 ENSEGLSRVY 487 A26 18 N.A. 102-110 LSRVYSKLY 488 A1 22 <1.0
[0378] 57 TABLE 34F SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Dinding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 92-125 101-110 GLSRVYSKLY 489 A1 18 <1.0 A26 18 N.A. A3 19 18 96-105 LENSEGLSRV 490 A0201 17 5 108-117 KLYKEAEKIK 491 A3 19 18 SCP-1 931-958 949-956 REDRWAVI 492 B5101 15 N.A. 948-956 MREDRWAVI 493 B2705 18 600 B2709 18 N.A. b5101 15 1 947-956 KMREDRWAVI 494 a0201 21 6 B08 N.A. 15 947-955 KMREDRWAV 495 A0201 22 411 934-942 TTPGSTLKF 496 A26 25 N.A. 933-942 LTTPGSTLKF 497 A26 23 N.A. 937-945 GSTLKFGAI 498 B08 19 1 945-953 IRKMREDRW 499 B08 19 <5 236-243 RLEMHFKL 500 B08 16 <5 SCP-1 232-259 235-243 SRLEMHFKL 501 A0201 18 <5 B2705 25 2000 B2709 22 242-250 KLKEDYEKI 502 A0201 4
[0379] 58 TABLE 34G SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 232-259 A26 16 N.A. (continued) A3 158 3 B08 24 <5 B5101 14 2 249-257 KIQHLEQEY 503 A1 15 <5 A26 23 N.A. A3 17 <5 248-257 EKIQHLEQEY 504 A1 15 <5 A26 21 N.A. 233-242 ENSRLEMHF 505 A26 19 N.A. 236-245 RLEMHFKLKE 506 A1 19 <5 A3 17 <5 324-331 LEDIKVSL 507 B08 20 <1.0 SCP-1 310-340 323-331 508 A0201 A26 25 N.A. A24 N.A. 10 A3 17 <1.0 B08 19 <1.0 B5101 16 N.A. 322-331 KELEDIKVSL 509 A0201 19 22 320-327 LTKELEDI 500 B08 18 <5 319-327 HLTKELEDI 511 A0201 21 <1.0 330-338 SLQRSVSTQ 512 A0201 18 <1.0
[0380] 59 TABLE 34H SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 310-340 321-329 TKELEDIKV 513 A1 16 <1.0 (continued) 320-329 LTKELEDIKV 514 A0201 19 <1.0 326-335 DIKVSLQRSV 515 A26 18 N.A. SCP-1 272-305 281-288 KMKDLTFL 516 B08 20 3 280-288 NKMKDLTFL 517 A0201 15 1 279-288 ENKMKDLTFL 518 A26 19 N.A. 288-296 LLEESRDKV 519 A0201 25 15 B5101 15 3 287-296 FLLEESRDKV 520 A0201 27 2378 291-299 ESRDKVNQL 521 A26 21 N.A. B08 29 240 290-299 EESRDKVNQL 522 A26 19 N.A. 277-285 EKENT(MKDL 523 A26 19 N.A. B08 23 <1.0 276-285 TEKENKMKDL 524 A26 15 N.A. 279-287 ENKMKDLTF 525 A26 18 N.A. B08 28 4 SCP-1 211-239 218-225 IEKIVHTAIF 526 B08 17 <5 217-225 NIEKMITAF 527 A26 26 N.A. 216-225 SNIEKMITAF 528 A26 19 N.A. 223-230 TAFEELRV 529 B5101 23 N.A. 222-230 LTAEEELRV 530 A0201 18 2 221-230 MITAFEELRV 531 A0201 18 16
[0381] 60 TABLE 34I SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Eptiope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 211-239 220-228 KMITAFEEL 532 A0201 23 50 (continued) A26 15 N.A. A24 N.A. 16 219-228 EKMITAEEEL 533 A26 19 N.A. 227-235 ELRVQAENS 534 A3 16 <1.0 B08 15 <1.0 213-222 DLNSNIEKMI 535 A0201 17 <1.0 A26 16 N.A. SCP-1 836-863 837-844 WTSAKNTL 536 B08 20 4 846-854 TPLPKAYTV 537 A0201 18 2 B0702 17 4 B08 16 2 B5101 25 220 845-854 STPLPKAYTV 538 A0201 19 <5 844-852 LSTPLPKAY 539 A1 23 8 843-852 TLSTPLPKAY 540 A1 16 <1.0 A26 19 N.A. A3 18 2 842-850 NTLSTPLPK 541 A3 16 3 841-850 KNTLSTPLPK 542 A3 18 <1.0
[0382] 61 TABLE 34J SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEIThI NIH SCP-1 819-845 828-835 ISKDKRDY 543 B08 21 3 A26 21 N.A. 826-835 HGISKDKRDY 544 A1 15 <5 832-840 KRDYLWTSA 545 B2 16 600 829-838 SKDKRDYLWT 546 A1 18 <5 SCP-1 260-288 279-286 ENKMKDLT 547 B08 22 8 260-268 EINDKEKQV 548 A0201 17 3 A26 19 N.A. B08 17 <5 274-282 QITEKENL 549 A0201 17 3 A26 22 N.A. B08 16 <5 269-277 SLLLIQITE 550 A0201 16 <1.0 A3 18 <1.0 SCP-1 437-464 453-460 FEKIAEEL 551 B08 21 <1.0 452-460 QFEKLAEEL 552 B2705 15 451-460 KQFEKIAEEL 553 A0201 16 56 449-456 DNKQFEKI 554 B08 16 2 B5101 16 N.A. 448-456 YDNKQFEKI 555 B5101 16 1 447-456 LYDNKQFEKI 556 A1 15 <1.0
[0383] 62 TABLE 34K SCP-1: Preferred Epitoopes Reealed by Housekeeping Proteasome Digestion Binding Prediction Substrate Epitope Sequence Seq. ID No. HLA type SYFPEITHI NIH SCP-1 437-464 440-447 LGEKETLL 557 B5101 16 N.A. (continued) 439-447 VLGEKETLL 558 A0201 24 149 A26 19 N.A. B08 29 12 438-447 KVLGEKETLL 559 A0201 19 24 A26 18 N.A. A24 N.A. 12 A3 18 <1.0 B0702 14 20 SCP-1 383-412 390-398 LLRTEQQRL 560 A0201 22 3 A26 18 N.A. B08 22 1.6 B2705 15 30 389-398 ELLRTEQQRL 561 A0201 19 6 A26 24 N.A. A3 15 <1.0 393-401 TEQQRLENY 562 A1 15 <5 A26 16 N.A. 392-401 RTEQQRLENY 563 A1 31 113 A26 26 N.A. 402-410 EDQLIILTM 564 A26 26 N.A. 397-406 RLENYEDQLI 565 A0201 17 <1.0 A3 15 <1.0
[0384] 63 TABLE 34L SCP-1: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Binding Prediction Seq. SYFP Sub- ID HLA EI strate Epitope Sequence No. type THI NIH SCP-1 368-375 KARAAHSF 566 B08 16 <1.0 366-394 376-384 VVTEFETTV 567 A0201 19 161 A3 16 <1.0 375-384 FVVTEFETTV 568 A0201 17 106 377-385 VTEFETTVC 569 A1 18 2 376-385 VVTEFETTVC 570 A3 16 <5 SCP-1 344-352 DLQIATNTI 571 A0201 22 <5 331-357 A3 15 <1.0 B5101 17 11 347-355 IATNTICQL 572 A0201 19 1 B08 16 <1.0 B5101 20 79 346-355 QIATNTICQL 573 A0201 24 7 A26 24 N.A.
[0385] 64 TABLE 35 SSX-4: Preferred Epitopes Revealed by Housekeeping Proteasome Digestion Seq. Binding Prediction Sub- ID SYFP strate Epitope Sequence No. HLA type EITHI NIH SSX4 57-65 VMTKLGFKV 574 A0201 21 495 45-76 53-61 LNYEVMTKL 575 A0201 17 7 52-61 KLNYEVMTKL 576 A0201 23 172 A26 21 N.A. A24 N.A. 18 A3 14 4 B7 N.A. 4 66-74 TLPPFMRSK 577 A26 16 N.A. A3 25 14 SSX4 110-118 KIMPKKPAE 578 A0201 15 <5 98-124 A26 15 N.A. A3 16 <5 103-112 SLQRIFPKIM 579 A0201 15 8 A26 16 N.A. A3 15 <5
[0386] 65 TABLE 36 Tyrosinase: Preferred Epitopes Revealed by House- keeping Proteasome Digestion Seq. Binding Prediction Sub- ID HLA SYFP strate Epitope Sequence No. type EITHI NIH Tyr 463-471 YIKSYLEQA 580 A0201 18 <5 445-474 A26 17 N.A. 459-467 SFQDYIKSY 581 A1 18 <5 A26 22 N.A. 458-467 DSFQDYIKSY 582 A1 19 <5 A26 24 N.A. Tyr 507-514 LPEEKQPL 583 B08 28 5 490-518 B5101 18 N.A. 506-514 QLPEEKQPL 584 A0201 22 88 A26 20 N.A. A24 N.A. 9 B08 18 <5 505-514 KQLPEEKQPL 585 A0201 15 28 A24 N.A. 17 507-515 LPEEKQPLL 586 A0201 15 <5 B0702 21 24 B08 28 5 B5101 21 157 506-515 QLPEEKQPLL 587 A0201 23 88 A26 20 N.A. A24 N.A. 7 497-505 SLLCHRHKRK 588 A3 25 15
Example 15[0387] Evaluating Likelihood of Epitope Cross-Reactivity on Non-Target Tissues.
[0388] As noted above PSA is a member of the kallikrein family of proteases, which is itself a subset of the serine protease family. While the members of this family sharing the greatest degree of sequence identity with PSA also share similar expression profiles, it remains possible that individual epitope sequences might be shared with proteins having distinctly different expression profiles. A first step in evaluating the likelihood of undesirable cross-reactivity is the identification of shared sequences. One way to accomplish this is to conduct a BLAST search of an epitope sequence against the SWISSPROT or Entrez non-redundant peptide sequence databases using the “Search for short nearly exact matches” option; hypertext transfer protocol accessible on the world wide web (http://www) at “ncbi.nlm.nih.gov/blast/index.html”. Thus searching SEQ ID NO. 214, WVLTAAHCI, against SWISSPROT (limited to entries for homo sapiens) one finds four exact matches, including PSA. The other three are from kallikrein 1 (tissue kallikrein), and elastase 2A and 2B. While these nine amino acid segments are identical, the flanking sequences are quite distinct, particularly on the C-terminal side, suggesting that processing may proceed differently and that thus the same epitope may not be liberated from these other proteins. (Please note that kallikrein naming is confused. Thus the kallikrein 1 [accession number P06870] is a different protein than the one [accession number AAD13817] mentioned in the paragraph on PSA above in the section on tumor-associated antigens).
[0389] It is possible to test this possibility in several ways. Synthetic peptides containing the epitope sequence embedded in the context of each of these proteins can be subjected to in vitro proteasomal digestion and analysis as described above. Alternatively, cells expressing these other proteins, whether by natural or recombinant expression, can be used as targets in a cytotoxicity (or similar) assay using CD8+ T cells that recognize the epitope, in order to determine if the epitope is processed and presented.
Example 16[0390] Epitope Clusters.
[0391] Known and predicted epitopes are generally not evenly distributed across the sequences of protein antigens. As referred to above, we have defined segments of sequence containing a higher than average density of (known or predicted) epitopes as epitope clusters. Among the uses of epitope clusters is the incorporation of their sequence into substrate peptides used in proteasomal digestion analysis as described herein. Epitope clusters can also be useful as vaccine components. A fuller discussion of the definition and uses of epitope clusters is found in U.S. patent application Ser. No. 09/561,571 entitled EPITOPE CLUSTERS, previously incorporated by reference in its entirety.
[0392] The following tables (37-60) present 9-mer epitopes predicted for HLA-A2 binding using both the SYFPEITHI and NIH algorithms and the epitope density of regions of overlapping eptiopes, and the epitopes in the whole protein, and the ratio of these two densities. (The ratio must exceed one to be a cluster by the above definition; requiring higher values of this ratio reflect preferred embodiments). Individual 9-mers are ranked by score and identified by the position of their first amino in the complete protein sequence. Each potential cluster from a protein is numbered. The range of amino acid positions within the complete sequence that the cluster cover is indicated as are the rankings of the individual predicted epitopes it is made up of. 66 TABLE 37 BIMAS-NIH/Parker algorithm Results for gp100 Rank Start Score 1 619 1493 2 602 413 3 162 226 4 18 118 5 178 118 6 273 117 7 601 81 8 243 63 9 606 60 10 373 50 11 544 36 12 291 29 13 592 29 14 268 29 15 47 27 16 585 26 17 576 21 18 465 21 19 570 20 20 9 19 21 416 19 22 25 18 23 566 17 24 603 15 25 384 14 26 13 14 27 290 12 28 637 10 29 639 9 30 485 9 31 453 8 32 102 8 33 399 8 34 456 7 35 113 7 36 622 7 37 69 7 38 604 6 39 350 6 40 583 5
[0393] 67 TABLE 38 SYFPEITHI (Rammensee algorithm) Results for gp100 Rank Start Score 1 606 30 2 162 29 3 456 28 4 18 28 5 602 27 6 598 27 7 601 26 8 597 26 9 13 26 10 585 25 11 449 25 12 4 25 13 603 24 14 576 24 15 453 24 16 178 24 17 171 24 18 11 24 19 619 23 20 280 23 21 268 23 22 592 22 23 544 22 24 465 22 25 399 22 26 373 22 27 273 22 28 243 22 29 566 21 30 563 21 31 485 21 32 384 21 33 350 21 34 9 21 35 463 20 36 397 20 37 291 20 38 269 20 39 2 20 40 610 19 41 594 19 42 591 19 43 583 19 44 570 19 45 488 19 46 446 19 47 322 19 48 267 19 49 250 19 50 205 19 51 180 19 52 169 19 53 88 19 54 47 19 55 10 19 56 648 18 57 605 18 58 604 18 59 595 18 60 571 18 61 569 18 62 450 18 63 409 18 64 400 18 65 371 18 66 343 18 67 298 18 68 209 18 69 102 18 70 97 18 71 76 18 72 69 18 73 60 18 74 17 18 75 613 17 76 599 17 77 572 17 78 557 17 79 556 17 80 512 17 81 406 17 82 324 17 83 290 17 84 101 17 85 95 17 86 635 16 87 588 16 88 584 16 89 577 16 90 559 16 91 539 16 92 494 16 93 482 16 94 468 16 95 442 16 96 413 16 97 408 16 98 402 16 99 286 16 100 234 16 101 217 16 102 211 16 103 176 16 104 107 16 105 96 16 106 80 16 107 16 16 108 14 16 109 7 16
[0394] 68 TABLE 39 Prediction of clusters for gp100 Total AAs: 661 Total 9-mers: 653 SYFPEITHI 16: 109 9-mers NIH 5:40 9-mers Epitopes/AA Cluster # AAs Epitopes (by Rank) Cluster Whole Pr Ratio SYFPEITHI 1 2 to 26 39, 12, 109, 34, 55, 11, 9, 0.440 0.165 2.668 108, 107, 74, 4 2 69—115 72, 71, 106, 53, 85, 105, 0.213 0.165 1.290 70, 84, 69, 104 3 95-115 85, 105, 70, 84, 69 0.238 0.165 1.444 4 162-188 2, 52, 17, 103, 16, 51 0.222 0.165 1.348 5 205-225 50, 68, 102, 101 0.190 0.165 1.155 6 243-258 28, 49 0.125 0.165 0.758 7 267-306 48, 21, 38, 27, 20, 99, 83, 37, 67 0.225 0.165 1.364 8 322-332 47, 82 0.182 0.165 1.103 9 343-358 66, 33 0.125 0.165 0.758 10 371-381 65, 26 0.182 0.165 1.103 11 397-421 36, 25, 64, 98, 81, 97, 63, 96 0.320 0.165 1.941 12 442-476 95, 46, 11, 62, 15, 3, 35, 24, 94 0.257 0.165 1.559 13 482-502 93, 31, 45, 93 0.190 0.165 1.155 14 539-552 91, 23 0.143 0.165 0.866 15 556-627 79, 78, 90, 30, 29, 61, 44, 60, 77, 14, 0.431 0.165 2.611 89, 43, 88, 10, 87, 42, 22, 41, 59, 8, 6, 76, 7, 5, 13, 58, 57, 1, 40, 75, 19 NIH 1 9 to 33 20, 26, 4, 22 0.160 0.061 2.644 2 268-281 14, 6 0.143 0.061 2.361 3 290-299 27, 12 0.200 0.061 3.305 4* 102-121 32, 35 0.100 0.061 1.653 5* 373-392 10, 25 0.100 0.061 1.653 6 453-473 31, 34, 18 0.143 0.061 2.361 7 566-600 23, 19, 17, 40, 16, 13 0.171 0.061 2.833 8 601-614 7, 2, 24, 38, 9 0.357 0.061 5.902 9 619-630 1, 36 0.17 0.061 2.754 10 637-647 28, 29 0.18 0.061 3.005 *Nearby but not overlapping epitopes
[0395] 69 TABLE 40 BIMAS-NIH/Parker algorithm Results for PSMA Rank Start Score 1 663 1360 2 711 1055 3 4 485 4 27 400 5 26 375 6 668 261 7 707 251 8 469 193 9 731 177 10 35 67 11 33 64 12 554 59 13 427 50 14 115 47 15 20 40 16 217 26 17 583 24 18 415 19 19 193 14 20 240 12 21 627 11 22 260 10 23 130 10 24 741 9 25 3 9 26 733 8 27 726 7 28 286 6 29 174 5 30 700 5
[0396] 70 TABLE 41 SYFPEITHI (Rammensee algorithm) Results for PSMA Rank Start Score 1 469 27 2 27 27 3 741 26 4 711 26 5 354 25 6 4 25 7 663 24 8 130 24 9 57 24 10 707 23 11 260 23 12 20 23 13 603 22 14 218 22 15 109 22 16 731 21 17 668 21 18 660 21 19 507 21 20 454 21 21 427 21 22 358 21 23 284 21 24 115 21 25 33 21 26 606 20 27 568 20 28 473 20 29 461 20 30 200 20 31 26 20 32 3 20 33 583 19 34 579 19 35 554 19 36 550 19 37 547 19 38 390 19 39 219 19 40 193 19 41 700 18 42 472 18 43 364 18 44 317 18 45 253 18 46 91 18 47 61 18 48 13 18 49 733 17 50 673 17 51 671 17 52 642 17 53 571 17 54 492 17 55 442 17 56 441 17 57 397 17 58 391 17 59 357 17 60 344 17 61 305 17 62 304 17 63 286 17 64 282 17 65 169 17 66 142 17 67 122 17 68 738 16 69 634 16 70 631 16 71 515 16 72 456 16 73 440 16 74 385 16 75 373 16 76 365 16 77 361 16 78 289 16 79 278 16 80 258 16 81 247 16 82 217 16 83 107 16 84 100 16 85 75 16 86 37 16 87 30 16 88 21 16
[0397] 71 TABLE 42 Prediction of clusters for prostate-specific membrane antigen (PSMA) Total AAs: 750 Total 9-mers: 742 SYFPEITHI 16: 88 9-mers NIH 5: 30 9-mers Epitopes/AA Cluster # Aas Epitopes (by rank) Cluster Whole Pr Ratio SYFPEITHI 1 3 to 12 32, 6 0.200 0.117 1.705 2 13-45 13, 12, 88, 31, 2, 87, 25, 86 0.242 0.117 2.066 3 57-69 9, 47 0.154 0.117 1.311 4 100-138 84, 83, 15, 24, 67, 8 0.154 0.117 1.311 5 193-208 40, 30 0.111 0.117 0.947 6 217-227 82, 14, 39 0.273 0.117 2.324 7 247-268 81, 45, 80, 11 0.182 0.117 1.550 8 278-297 79, 64, 23, 63, 78 0.250 0.117 2.131 9 354-381 5, 59, 22, 77, 43, 76, 75 0.250 0.117 2.131 10 385-405 74, 38, 58, 57 0.190 0.117 1.623 11 440-450 73, 56, 55 0.273 0.117 2.324 12 454-481 20, 72, 29, 1, 42, 28 0.214 0.117 1.826 13 507-523 17, 71 0.118 0.117 1.003 14 547-562 37, 36, 35 0.188 0.117 1.598 15 568-591 27, 53, 34, 33 0.167 0.117 1.420 16 603-614 13, 26 0.167 0.117 1.420 17 631-650 70, 69, 52 0.150 0.117 1.278 18 660-681 18, 7, 17, 51, 50 0.227 0.117 1.937 19 700-719 41, 10, 4 0.150 0.117 1.278 20 731-749 16, 49, 68, 3 0.211 0.117 1.794 NIH 1 3 to 12 25, 3 0.200 0.040 5.000 2 20-43 15, 5, 4, 11, 10 0.208 0.040 5.208 3* 415-435 18, 13 0.095 0.040 2.381 4 663-676 1, 6 0.143 0.040 3.571 5 700-715 30, 7, 3 0.188 0.040 4.688 6 726-749 27, 9, 26, 24 0.167 0.040 4.167 *Nearby but not overlapping epitopes
[0398] 72 TABLE 43 BIMAS-NIH/Parker algorithm Results for PSA Rank Start Score 1 7 607 2 170 243 3 52 124 4 53 112 5 195 101 6 165 23 7 72 18 8 245 18 9 2 16 10 59 16 11 122 15 12 125 15 13 191 13 14 9 8 15 14 6 16 175 5 17 130 5
[0399] 73 TABLE 44 SYFPEITHI (Rammensee algorithm) Results for PSA Rank Start Score 1 72 26 2 170 22 3 53 22 4 7 22 5 234 21 6 166 21 7 140 21 8 66 21 9 241 20 10 175 20 11 12 20 12 41 19 13 20 19 14 14 19 15 130 18 16 124 18 17 121 18 18 47 18 19 17 18 20 218 17 21 133 17 22 125 17 23 122 17 24 118 17 25 110 17 26 67 17 27 52 17 28 21 17 29 16 17 30 2 17 31 184 16 32 179 16 33 158 16 34 79 16 35 73 16 36 4 16
[0400] 74 TABLE 45 Prediction of clusters for prostate specific antigen (PSA) Total AAs: 261 Total 9-mers: 253 SYFPEITHI 16: 36 9-mers NIH 5: 17 9-mers Epitopes/AA Cluster # AAs Epitopes (by rank) Cluster Whole Pr Ratio SYFPEITHI 1 2 to 29 30, 36, 4, 11, 14, 29, 19, 13, 28 0.321 0.138 2.330 2 41-61 12, 18, 27, 3 0.190 0.138 1.381 3 66-87 8, 26, 1, 35, 34 0.227 0.138 1.648 4 110-148 25, 24, 17, 23, 16, 22, 15, 21, 7 0.184 0.138 1.332 5 158-192 33, 6, 2, 10, 32, 31 0.171 0.138 1.243 6 234-249 5, 9 0.125 0.138 0.906 7* 118-133 24, 17, 23, 16, 22 0.313 0.138 2.266 8* 118-138 24, 17, 23, 16, 22, 15 0.286 0.138 2.071 NIH 1 2-22 9, 1, 14, 15 0.190 0.065 2.924 2 52-67 3, 4, 10 0.188 0.065 2.879 3 122-138 11, 12, 17 0.176 0.065 2.709 4 165-183 6, 2, 16 0.158 0.065 2.424 5 191-203 13, 5 0.154 0.065 2.362 6** 52-80 3, 4, 10, 7 0.138 0.065 2.118 *These clusters are internal to the less preferred cluster #4. **Includes a nearby but not overlapping epitope.
[0401] 75 TABLE 46 BIMAS-NIH/Parker algorithm Results for PSCA Rank Start Score 1 43 153 2 5 84 3 7 79 4 109 36 5 105 25 6 108 24 7 14 21 8 20 18 9 115 17 10 42 15 11 36 15 12 99 9 13 58 8
[0402] 76 TABLE 47 SYFPEITHI (Rammensee algorithm) Results for PSCA Rank Start Score 1 108 30 2 14 30 3 105 29 4 5 28 5 115 26 6 99 26 7 7 26 8 109 24 9 53 23 10 107 21 11 20 21 12 8 21 13 13 20 14 102 19 15 60 19 16 57 19 17 54 19 18 12 19 19 4 19 20 1 19 21 112 18 22 101 18 23 98 18 24 51 18 25 43 18 26 106 17 27 104 17 28 83 17 29 63 17 30 50 17 31 3 17 32 9 16 33 92 16
[0403] 77 TABLE 48 Prediction of clusters for prostate stem cell antigen (PSCA) Total AAs: 123 Total 9-mers: 115 SYFPEITHI 16: 33; SYFPEITHI 20: 13 NIH 5: 13 Epitopes/AA Cluster # AAs Epitopes (by rank) Cluster Whole Pr. Ratio SYFPEITHI>16 1 1 to 28 20, 31, 19, 4, 7, 12, 33, 18, 13, 2, 11 0.393 0.268 1.464 2 43-71 25, 30, 24, 9, 17, 16, 15, 29 0.276 0.268 1.028 3 92-123 32, 23, 6, 27, 14, 22, 3, 26, 10, 0.406 0.268 1.514 1, 8, 21, 5 SYFPEITHI >20 1 5 to 28 4, 7, 12, 13, 2, 11 0.250 0.106 2.365 2 99-123 6, 3, 10, 1, 8, 5 0.240 0.106 2.271 NIH 1 5 to 28 2, 3, 7, 8 0.167 0.106 1.577 2 36-51 11, 10, 1 0.188 0.106 1.774 3 99-123 12, 5, 6, 4, 9 0.200 0.106 1.892 4* 105-116 5, 6, 4 0.250 0.106 2.365 *This cluster is internal to the less preferred cluster #3.
[0404] In tables 49-60 epitope prediction and cluster analysis data for each algorithm are presented together in a single table. 78 TABLE 49 Prediction of clusters for MAGE-1 (NIH algorithm) Total AAs: 309 Total 9-mers: 301 NIH 5: 19 9-mers Start Epitopes/AA Epitope Posi- NIH Whole Cluster # AAs Rank tion Score Cluster Pr. Ratio 1 18-32 16 18 9 0.133 0.063 2.112 19 24 7 2 101-113 14 101 11 0.154 0.063 2.442 7 105 44 3 146-159 9 146 32 0.143 0.063 2.263 3 151 169 4 169-202 10 169 32 0.176 0.063 2.796 13 174 16 18 181 8 17 187 8 6 188 74 5 194 110 5 264-277 2 264 190 0.143 0.063 2.263 12 269 20 6 278-290 1 278 743 0.154 0.063 2.437 11 282 28
[0405] 79 TABLE 50 Prediction of clusters for MAGE-1 (SYFPEITHI algorithm) Epitope Start SYFPEITHI Epitopes/AA Cluster # Aas Rank Position Score Cluster Whole Ratio 1 7-49 22 7 19 0.233 0.153 1.522 9 15 22 27 18 18 16 20 20 28 22 18 29 24 18 33 31 17 30 35 18 2 38 26 17 41 20 2 89-132 10 89 22 0.273 0.153 1.783 18 92 20 7 93 23 23 96 19 43 98 16 4 101 25 8 105 23 34 107 17 35 108 17 36 113 17 37 118 17 19 124 20 3 167-203 44 167 16 0.270 0.153 1.766 20 169 20 12 174 21 24 181 19 6 187 24 31 188 18 25 191 19 38 192 17 1 194 27 13 195 21 4 230-246 14 230 21 0.118 0.153 0.769 39 238 17 5 264-297 15 264 21 0.235 0.153 1.538 32 269 18 40 270 17 26 271 19 46 275 16 3 278 26 21 282 20 41 289 17 Total AAs: 309 Total 9-mers: 301 SYFPEITHI 16: 46 9-mers
[0406] 80 TABLE 51 Prediction of clusters for MAGE-2 (NIH algorithm) Epitope Start NIH Epitope/AA Cluster # AAs Rank Position Score Cluster Whole Pr. Ratio 1 101-120 18 101 5.373 0.150 0.065 2.310 16 108 6.756 1 112 2800.697 2 153-167 8 153 31.883 0.200 0.065 3.080 4 158 168.552 7 159 32.138 3 169-211 14 169 8.535 0.209 0.065 3.223 19 174 5.346 6 176 49.993 11 181 15.701 15 188 7.536 12 195 12.809 5 200 88.783 10 201 16.725 17 203 5.609 4 271-284 3 271 398.324 0.143 0.065 2.200 9 276 19.658 Total AAs: 314 Total 9-mers: 308 NIH >= 5: 20 9-mers
[0407] 81 TABLE 52 Prediction of clusters for MAGE-2 (SYFPEITHI algorithm) Epitope Start SYFPEITHI Epitopes/AA Cluster # AAs Rank Position Score Cluster Whole Pr. Ratio 1 15-32 13 15 21 0.278 0.169 1.645 29 18 18 43 20 16 30 22 18 21 24 19 2 37-56 31 37 18 0.250 0.169 1.481 16 40 20 44 44 16 14 45 21 22 48 19 3 96-133 36 96 17 0.211 0.169 1.247 46 101 16 6 108 25 47 109 16 2 112 27 37 120 17 38 125 17 17 131 20 4 153-216 12 153 22 0.344 0.169 2.036 39 158 17 7 159 25 23 161 19 24 162 19 48 164 16 49 167 16 32 170 18 50 171 16 4 174 26 9 176 24 51 177 16 15 181 21 25 188 19 18 194 20 33 195 18 19 198 20 3 200 27 1 201 28 40 202 17 10 203 23 52 208 16 5 237-254 26 237 19 0.167 0.169 0.987 27 245 19 34 246 18 6 271-299 8 271 25 0.241 0.169 1.430 35 276 18 41 277 17 11 278 23 28 283 19 20 285 20 42 291 17 Total AAs: 314 Total 9-mers: 308 SYFPEITHI 16: 52 9-mers
[0408] 82 TABLE 53 Prediction of clusters for MAGE-3 (NIH algorithm) Epitope Start NIH Epitopes/AA Cluster # AAs Rank Position Score Cluster Whole Pr. Ratio 1 101-120 15 101 11.002 0.200 0.071 2.800 21 105 6.488 8 108 49.134 2 112 339.313 2 153-167 18 153 7.776 0.200 0.071 2.800 6 158 51.77 22 159 5.599 3 174-209 17 174 8.832 0.194 0.071 2.722 7 176 49.993 13 181 15.701 19 188 7.536 14 195 12.809 5 200 88.783 12 201 16.725 4 237-251 16 237 10.868 0.200 0.071 2.800 4 238 148.896 20 243 6.88 5 271-284 1 271 2655.495 0.143 0.071 2.000 11 276 19.658 Total AAs: 314 Total 9-mers: 308 NIH 5: 22 9-mers
[0409] 83 TABLE 54 Prediction of clusters for MAGE-3 (SYFPEITHI algorithm) Epitope Start SYFPEITHI Epitopes/AA Cluster # AAs Rank Position Score Cluster Whole Pr. Ratio 1 15-32 12 15 21 0.278 0.153 1.820 26 18 18 37 20 16 27 22 18 18 24 19 2 38-56 38 38 16 0.263 0.153 1.725 15 40 20 39 44 16 13 45 21 19 48 19 3 101-142 28 101 18 0.190 0.153 1.248 40 105 16 1 108 31 6 112 25 31 120 17 32 125 17 16 131 20 41 134 16 4 153-216 20 153 19 0.313 0.153 2.048 29 156 18 33 158 17 21 159 19 34 161 17 42 164 16 43 167 16 10 174 22 8 176 23 14 181 21 22 188 19 44 193 16 11 194 22 23 195 19 45 197 16 17 198 20 3 200 27 2 201 28 35 202 17 46 208 16 5 220-230 5 220 26 0.182 0.153 1.191 47 222 16 6 237-246 7 237 25 0.200 0.153 1.311 9 238 23 7 271-293 4 271 27 0.217 0.153 1.425 30 276 18 24 278 19 36 283 17 25 285 19 Total AAs: 314 Total 9-mers: 308 SYFPEITHI 16: 47 9-mers
[0410] 84 TABLE 55 Prediction of clusters for PRAME (NIH algorithm) Epitope Start NIH Epitopes/AA Cluster # AAs Rank Position Score Cluster Whole Pr. Ratio 1 33-47 20 33 18 0.133 0.080 1.670 17 39 21 2 71-81 9 71 50 0.2 0.07984 2.505 32 73 7 3 99-108 23 100 15 0.2 0.07984 2.505 24 99 13 4 126-135 38 126 5 0.2 0.07984 2.505 35 127 6 5 224-246 5 224 124 0.130 0.080 1.634 8 230 63 39 238 5 6 290-303 18 290 18 0.214 0.080 2.684 14 292 23 7 295 66 7 305-324 28 305 10 0.200 0.080 2.505 30 308 8 25 312 13 36 316 6 8 394-409 2 394 182 0.188 0.080 2.348 12 397 42 31 401 7 9 422-443 10 422 49 0.227 0.080 2.847 3 425 182 34 431 7 29 432 9 4 435 160 10 459-487 15 459 21 0.172 0.080 2.159 11 462 45 22 466 15 40 472 5 37 479 6 Total AAs: 509 Total 9-mers: 501 NIH 5: 40 9-mers
[0411] 85 TABLE 56 Prediction of clusters for PRAME (SYFPEITHI algorithm) Epitope Start SYFPEITHI Epitopes/AA Cluster # AAs Rank Position Score Cluster Whole Pr. Ratio 1 18-59 65 18 17 0.238 0.160 1.491 50 21 18 66 26 17 35 33 20 22 34 22 51 37 18 5 39 27 23 40 22 13 44 24 46 51 19 2 78-115 36 78 20 0.263 0.160 1.648 67 80 17 52 84 18 24 86 22 53 91 18 25 93 22 9 99 25 8 100 26 54 103 18 55 107 18 3 191-202 56 191 18 0.167 0.160 1.044 38 194 20 4 205-215 26 205 22 0.182 0.160 1.139 27 207 22 5 222-238 47 222 19 0.235 0.160 1.474 14 224 24 69 227 17 57 230 18 6 241-273 70 241 17 0.212 0.160 1.328 15 248 24 71 255 17 30 258 21 39 259 20 58 261 18 40 265 20 7 290-342 72 290 17 0.208 0.160 1.300 48 293 19 31 298 21 73 301 17 18 305 23 6 308 27 10 312 25 19 316 23 28 319 22 41 326 20 74 334 17 8 343-363 59 343 18 0.238 0.160 1.491 60 348 18 75 351 17 20 353 23 76 355 17 9 364-447 49 364 19 0.250 0.160 1.566 32 371 21 11 372 25 61 375 18 77 382 17 21 390 23 78 391 17 1 394 30 42 397 20 62 403 18 33 410 21 43 418 20 34 419 21 7 422 27 2 425 29 79 426 17 63 428 18 64 431 18 12 432 25 16 435 24 80 439 17 10 455-474 29 455 22 0.200 0.160 1.253 17 459 24 4 462 28 3 466 29 Total AAs: 509 Total 9-mers: 501 SYFPEITHI 17: 80 9-mers
[0412] 86 TABLE 57 Predication of clusters for CEA (NIH algorithm) Cluster Peptides Start Peptides/AAs # AA Rank Position Score Cluster Whole Pr. Ratio 1 17-32 5 17 79.041 0.188 0.043 4.388 7 18 46.873 20 24 12.668 2 113-129 2 113 167.991 0.118 0.043 2.753 15 121 21.362 3 172-187 25 172 9.165 0.125 0.043 2.925 14 179 27.995 4 278-219 30 278 5.818 0.143 0.043 3.343 17 283 19.301 5 350-365 9 350 43.075 0.125 0.043 2.925 12 357 27.995 6 528-543 8 528 43.075 0.125 0.043 2.925 13 535 27.995 7 631-645 23 631 9.563 0.200 0.043 4.680 19 634 13.381 24 637 9.245 8 691-702 1 691 196.407 0.167 0.043 3.900 27 694 7.769 Total AAs: 702 Total 9-mers: 694 NIH 5: 30 9-mers
[0413] 87 TABLE 58 Predication of clusters for CEA (SYFPEITHI algorithm) Peptides/AAs Cluster Peptides Start Whole # AA Rank Position Score Cluster Pr. Ratio 1 5-36 67 5 16 0.250 0.117 2.140 23 12 19 24 16 19 9 17 22 25 18 19 32 19 18 68 23 16 33 28 18 2 37-62 41 37 17 0.269 0.117 2.305 20 44 20 26 45 19 42 46 17 27 50 19 43 53 17 44 54 17 3 99-115 14 99 21 0.235 0.117 2.014 5 100 23 45 104 17 34 107 18 4 116-129 69 116 16 0.143 0.117 1.223 21 121 20 5 172-187 46 172 17 0.125 0.117 1.070 70 179 16 6 192-202 3 192 24 0.182 0.117 1.557 47 194 17 7 226-241 48 226 17 0.188 0.117 1.605 49 229 17 15 233 21 8 307-318 11 307 22 0.250 0.117 2.140 71 308 16 51 310 17 9 319-349 52 319 17 0.129 0.117 1.105 53 327 17 72 335 16 35 341 18 10 370-388 12 370 22 0.211 0.117 1.802 54 372 17 74 375 16 6 380 23 11 403-419 56 403 17 0.235 0.117 2.014 57 404 17 58 407 17 28 411 19 12 427-442 59 427 17 0.188 0.117 1.605 75 432 16 76 434 16 13 450-462 77 450 16 0.154 0.117 1.317 13 454 22 14 488-505 36 488 18 0.167 0.117 1.427 18 492 21 60 497 17 15 548-558 4 548 24 0.182 0.117 1.557 61 550 17 16 565-577 62 565 17 0.154 0.117 1.317 19 569 21 17 579-597 78 579 16 0.143 0.117 1.223 79 582 16 7 589 23 18 605-618 2 605 25 0.143 0.117 1.223 38 610 18 19 631-669 29 631 19 0.154 0.117 1.317 63 637 17 80 644 16 64 652 17 39 660 18 81 661 16 20 675-702 22 675 20 0.286 0.117 2.446 30 683 19 31 687 19 40 688 18 65 690 17 1 691 31 66 692 17 8 694 23 Total AAs: 702 Total 9-mers: 694 SYFPEITHI 16: 81 9-mers
[0414] 88 TABLE 59 Predication of clusters for SCP-1 (NIH algorithm) Pep- Start Peptides/ Cluster tides Posi- AAs # AA Rank tion Score Cluster Whole Pr. Ratio 1 101-116 15 101 40.589 0.125 0.038 3.270 13 108 57.255 2* 281-305 14 281 44.944 0.12 0.038 3.139 24 288 15.203 17 297 32.857 3 431-447 8 431 80.217 0.073 0.038 1.914 26 438 11.861 4 439 148.896 4 557-579 11 557 64.335 0.174 0.038 4.550 19 560 24.937 6 564 87.586 18 571 32.765 5 635-650 10 635 69.552 0.125 0.038 3.270 34 642 6.542 6 755-767 36 755 5.599 0.154 0.038 4.025 35 759 5.928 7 838-854 2 838 284.517 0.118 0.038 3.078 28 846 11.426 Total AAs: 976 Total 9-mers: 968 NIH 5: 37 9-mers
[0415] 89 TABLE 60 Predication of clusters for SCP-1 Pep- Start Peptides/ Cluster tides Posi- AAs # AA Rank tion Score Cluster Whole Pr. Ratio 1 8-28 99 8 16 0.143 0.121 1.182 77 15 17 100 20 16 2 63-80 78 63 17 0.222 0.121 1.838 50 66 19 102 69 16 60 72 18 3 94-123 79 94 17 0.133 0.121 1.103 12 101 23 17 108 22 103 115 16 4 126-158 35 126 20 0.182 0.121 1.504 36 133 20 51 139 19 80 140 17 61 143 18 37 150 20 5 161-189 38 161 20 0.207 0.121 1.711 52 165 19 81 171 17 82 177 17 62 178 18 39 181 20 6 213-230 40 213 20 0.167 0.121 1.379 13 220 23 28 222 21 7 235-250 63 235 18 0.125 0.121 1.034 18 242 22 8 260-296 83 260 17 0.243 0.121 2.012 105 262 16 84 267 17 106 269 16 41 270 20 64 271 18 85 274 17 19 281 22 3 288 25 9 312-338 108 312 16 0.148 0.121 1.225 29 319 21 30 323 21 65 330 18 10 339-355 66 339 18 0.235 0.121 1.946 31 340 21 42 344 20 53 347 19 11 376-447 54 376 19 0.194 0.121 1.608 43 382 20 44 386 20 20 390 22 55 397 19 6 404 24 86 407 17 45 411 20 67 417 18 21 425 22 46 431 20 68 432 18 32 438 21 7 439 24 12 455-488 33 455 21 0.235 0.121 1.946 47 459 20 56 462 19 87 463 17 88 466 17 14 470 23 109 473 16 34 480 21 13 515-530 57 515 19 0.125 0.121 1.034 22 522 22 14 557-590 8 557 24 0.147 0.121 1.216 23 564 22 9 571 24 90 575 17 58 582 19 15 610-625 69 610 18 0.125 0.121 1.034 91 617 17 16 633-668 92 633 17 0.222 10 635 24 70 638 18 93 640 17 48 642 20 49 645 20 111 652 16 112 660 16 17 674-685 71 674 18 0.167 0.121 1.379 11 677 24 18 687-702 1 687 26 0.125 0.121 1.034 94 694 17 19 744-767 113 744 16 0.250 0.121 2.068 95 745 17 4 745 25 24 752 22 2 755 26 72 759 18 20 812-827 97 812 17 0.125 0.121 1.034 115 819 16 21 838-857 116 838 16 0.150 0.121 1.241 25 846 22 74 849 18 22 896-913 117 896 16 0.222 0.121 1.838 98 899 17 26 902 22 76 905 18 Total AAs: 976 Total 9-mers: 968 Rammensee 16: 118 9-mers
[0416] The embodiments of the invention are applicable to and contemplate variations in the sequences of the target antigens provided herein, including those disclosed in the various databases that are accessible by the world wide web. Specifically for the specific sequences disclosed herein, variation in sequences can be found by using the provided accession numbers to access information for each antigen. 90 TYROSINASE PROTEIN; SEQ ID NO 2 1 MLLAVLYCLL WSFQTSAGHF PRACVSSKNL MEKECCPPWS GDRSPCGQLS GRGS CQNILL 61 SNAPLGPQFP FTGVDDRESW PSVFYNRTCQ CSGNFMGFNC GNCKFGFWGP NCTERRLLVR 121 RNIFDLSAPE KDKFFAYLTL AKHTISSDYV IPIGTYGQMK NGSTPMFNDI NIYDLFVWMH 181 YYVSMDALLG GSEIWRDIDF AHEAPAFLPW HRLFLLRWEQ EIQKLTGDEN FTIPYWDWRD 241 AEKCDICTDE YMGGQHPTNP NLLSPASFFS SWQIVCSRLE EYNSHQSLCN GTPEGPLRRN 301 PGNHDKSRTP RLPSSADVEF CLSLTQYESG SMDKAANFSF RNTLEGFASP LTGIADASQS 361 SMHNALHIYM NGTMSQVQGS ANDPIFLLHH AFVDSIFEQW LRRHRPLQEV YPEANAPIGH 421 NRESYMVPFI PLYRNGDFFI SSKDLGYDYS YLQDSDPDSF QDYIKSYLEQ ASRIWSWLLG 481 AAMVGAVLTA LLAGLVSLLC RHKRKQLPEE KQPLLMEKED YHSLYQSHL SSX-2 PROTEIN; SEQ ID NO 3 1 MNGDDAFARR PTVGAQIPEK IQKAFDDIAK YFSKEEWEKM KASEKIFYVY MKRKYEAMTK 61 LGFKATLPPF MCNKRAEDFQ GNDLDNDPNR GNQVERPQMT FGRLQGISPK IMPKKPAEEG 121 NDSEEVPEAS GPQNDGKELC PPGKPTTSEK IHERSGPKRG EHAWTHRLRE RKQLVIYEEI 181 SDPEEDDE PSMA PROTEIN; SEQ ID NO 4 1 MWNLLHETDS AVATARRPRW LCAGALVLAG GFFLLGFLFG WFIKSSNEAT NITPKHNMKA 61 FLDELKAENI KKFLYNFTQI PHLAGTEQNF QLAKQIQSQW KEFGLDSVEL AHYDVLLSYP 121 NKTHPNYISI INEDGNEIFN TSLFEPPPPG YENVSDIVPP FSAFSPQGMP EGDLVYVNYA 181 RTEDFFKLER DMKINCSGKI VIARYGKVFR GNKVKNAQLA GAKGVILYSD PADYFAPGVK 241 SYPDGWNLPG GGVQRGNILN LNGAGDPLTP GYPANEYAYR RGIAEAVGLP SIPVHPIGYY 301 DAQKLLEKMG GSAPPDSSWR GSLKVPYNVG PGFTGNFSTQ KVKMHIHSTN EVTRIYNVIG 361 TLRGAVEPDR YVILGGHRDS WVFGGIDPQS GAAVVHEIVR SFGTLKKEGW RPRRTILFAS 421 WDAEEFGLLG STEWAEENSR LLQERGVAYI NADSSIEGNY TLRVDCTPLM YSLVHNLTKE 481 LKSPDEGFEG KSLYESWTKK SPSPEFSGMP RISKLGSGND FEVFFQRLGI ASGRARYTKN 541 WETNKFSGYP LYHSVYETYE LVEKFYDPMF KYHLTVAQVR GGMVFELANS IVLPFDCRDY 601 AVVLRKYADK IYSISMKHPQ EMKTYSVSFD SLFSAVKNFT EIASKFSERL QDFDKSNPIV 661 LRMMNDQLMF LERAFIDPLG LPDRPFYRHV IYAPSSHNKY AGESFPGIYD ALFDIESKVD 721 PSKAWGEVKR QIYVAAFTVQ AAAETLSEVA Homo sapiens tyrosinase (oculocutaneous albinism IA) (TYR), mRNA.; ACCESSION NM_000372 VERSION NM_000372.1 GI:4507752 SEQ ID NO 2 /translation = “MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRS PCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGN CKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMK NGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLLRW EQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSWQIVC SRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDK AANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAF VDSIFEQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGYDYS YLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCRHKRKQLP EEKQPLLMEKEDYHSLYQSHL” SEQ ID NO 5 ORIGIN 1 atcactgtag tagtagctgg aaagagaaat ctgtgactcc aattagccag ttcctgcaga 61 ccttgtgagg actagaggaa gaatgctcct ggctgttttg tactgcctgc tgtggagttt 121 ccagacctcc gctggccatt tccctagagc ctgtgtctcc tctaagaacc tgatggagaa 181 ggaatgctgt ccaccgtgga gcggggacag gagtccctgt ggccagcttt caggcagagg 241 ttcctgtcag aatatccttc tgtccaatgc accacttggg cctcaatttc ccttcacagg 301 ggtggatgac cgggagtcgt ggccttccgt cttttataat aggacctgcc agtgctctgg 361 caacttcatg ggattcaact gtggaaactg caagtttggc ttttggggac caaactgcac 421 agagagacga ctcttggtga gaagaaacat cttcgatttg agtgccccag agaaggacaa 481 attttttgcc tacctcactt tagcaaagca taccatcagc tcagactatg tcatccccat 541 agggacctat ggccaaatga aaaatggatc aacacccatg tttaacgaca tcaatattta 601 tgacctcttt gtctggatgc attattatgt gtcaatggat gcactgcttg ggggatctga 661 aatctggaga gacattgatt ttgcccatga agcaccagct tttctgcctt ggcatagact 721 cttcttgttg cggtgggaac aagaaatcca gaagctgaca ggagatgaaa acttcactat 781 tccatattgg gactggcggg atgcagaaaa gtgtgacatt tgcacagatg agtacatggg 841 aggtcagcac cccacaaatc ctaacttact cagcccagca tcattcttct cctcttggca 901 gattgtctgt agccgattgg aggagtacaa cagccatcag tctttatgca atggaacgcc 961 cgagggacct ttacggcgta atcctggaaa ccatgacaaa tccagaaccc caaggctccc 1021 ctcttcagct gatgtagaat tttgcctgag tttgacccaa tatgaatctg gttccatgga 1081 taaagctgcc aatttcagct ttagaaatac actggaagga tttgctagtc cacttactgg 1141 gatagcggat gcctctcaaa gcagcatgca caatgccttg cacatctata tgaatggaac 1201 aatgtcccag gtacagggat ctgccaacga tcctatcttc cttcttcacc atgcatttgt 1261 tgacagtatt tttgagcagt ggctccgaag gcaccgtcct cttcaagaag tttatccaga 1321 agccaatgca cccattggac ataaccggga atcctacatg gttcctttta taccactgta 1381 cagaaatggt gatttcttta tttcatccaa agatctgggc tatgactata gctatctaca 1441 agattcagac ccagactctt ttcaagacta cattaagtcc tatttggaac aagcgagtcg 1501 gatctggtca tggctccttg gggcggcgat ggtaggggcc gtcctcactg ccctgctggc 1561 agggcttgtg agcttgctgt gtcgtcacaa gagaaagcag cttcctgaag aaaagcagcc 1621 actcctcatg gagaaagagg attaccacag cttgtatcag agccatttat aaaaggctta 1681 ggcaatagag tagggccaaa aagcctgacc tcactctaac tcaaagtaat gtccaggttc 1741 ccagagaata tctgctggta tttttctgta aagaccattt gcaaaattgt aacctaatac 1801 aaagtgtagc cttcttccaa ctcaggtaga acacacctgt ctttgtcttg ctgttttcac 1861 tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatg ctatttggta 1921 atgaggaact gttatttgta tgtgaattaa agtgctctta tttt Homo sapiens synovial sarcoma, X breakpoint 2 (SSX2), mRNA. ACCESSION NM_003147 VERSION NM_003147.1 GI:10337582 SEQ ID NO 3 /translation = “MNGDDAFARRPTVGAQIPEKIQKAFDDIAKYFSKEEWEKMKASE KIFYVYMKRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNRGNQVERPQMTFG RLQGISPKIMPKKPAEEGNDSEEVPEASGPQNDGKELCPPGKPTTSEKIHERSGPKRG EHAWTHRLRERKQLVIYEEISDPEEDDE” SEQ ID NO 6 ORIGIN 1 ctctctttcg attcttccat actcagagta cgcacggtct gattttctct ttggattctt 61 ccaaaatcag agtcagactg ctcccggtgc catgaacgga gacgacgcct ttgcaaggag 121 acccacggtt ggtgctcaaa taccagagaa gatccaaaag gccttcgatg atattgccaa 181 atacttctct aaggaagagt gggaaaagat gaaagcctcg gagaaaatct tctatgtgta 241 tatgaagaga aagtatgagg ctatgactaa actaggtttc aaggccaccc tcccaccttt 301 catgtgtaat aaacgggccg aagacttcca ggggaatgat ttggataatg accctaaccg 361 tgggaatcag gttgaacgtc ctcagatgac tttcggcagg ctccagggaa tctccccgaa 421 gatcatgccc aagaagccag cagaggaagg aaatgattcg gaggaagtgc cagaagcatc 481 tggcccacaa aatgatggga aagagctgtg ccccccggga aaaccaacta cctctgagaa 541 gattcacgag agatctggac ccaaaagggg ggaacatgcc tggacccaca gactgcgtga 601 gagaaaacag ctggtgattt atgaagagat cagcgaccct gaggaagatg acgagtaact 661 cccctcaggg atacgacaca tgcccatgat gagaagcaga acgtggtgac ctttcacgaa 721 catgggcatg gctgcggacc cctcgtcatc aggtgcatag caagtg Homo sapiens folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1), mRNA. ACCESSION NM_004476 VERSION NM_004476.1 GI:4758397 /translation = “MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIK SSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKE FGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDTVPP FSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQ LAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANE YAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFT GNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGA AVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSG MPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFY DPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKT YSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLP DRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQ AAAETLSEVA” SEQ ID NO 7 ORIGIN 1 ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg 61 attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga 121 gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac 181 cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag 241 gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc 301 accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt 361 ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctac taacattact 421 ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc 481 ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca 541 aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat 601 gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa 661 gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccagg atatgaaaat 721 gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat 781 ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa 841 atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag 901 gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac 961 tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg aggtggtgtc 1021 cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacacc aggttaccca 1081 gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1141 gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca 1201 ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt 1261 actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca 1321 agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag atatgtcatt 1381 ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct 1441 gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga 1501 agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag 1561 tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac 1621 tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg 1681 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt 1741 tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc 1801 aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat tgcttcaggc 1861 agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac 1921 agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac 1981 ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc 2041 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt 2101 atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga ttcacttttt 2161 tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagact ccaggacttt 2221 gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga 2281 gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct 2341 ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt 2401 gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag acagatttat 2461 gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagc ctaagaggat 2521 tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2581 atattgataa attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa 2641 aaaaaaaaaa aaa Human melanocyte-specific (pmel 17) gene, exons 2-5, and complete cds. ACCESSION U20093 VERSION U20093.1 GI:1142634 SEQ ID NO 70 /translation = “MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKWNRQLYPE WTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVW GGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGT HTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHD PSGYLAEADLSYTWDFGDSSGTLISRAPVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTD GHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTP EKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDA SSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGE GDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSL ADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSH WLRLPRIFCSCPIGENSPLLSGQQV” SEQ ID NO 80 ORIGIN 1 gtgctaaaaa gatgccttct tcatttggct gtgataggtg ctttgtggct gtgggggcta 61 caaaagtacc cagaaaccag gactggcttg gtgtctcaag gcaactcaga accaaagcct 121 ggaacaggca gctgtatcca gagtggacag aagcccagag acttgactgc tggagaggtg 181 gtcaagtgtc cctcaaggtc agtaatgatg ggcctacact gattggtgca aatgcctcct 241 tctctattgc cttgaacttc cctggaagcc aaaaggtatt gccagatggg caggttatct 301 gggtcaacaa taccatcatc aatgggagcc aggtgtgggg aggacagcca gtgtatcccc 361 aggaaactga cgatgcctgc atcttccctg atggtggacc ttgcccatct ggctcttggt 421 ctcagaagag aagctttgtt tatgtctgga agacctgggg tgagggactc ccttctcagc 481 ctatcatcca cacttgtgtt tacttctttc tacctgatca cctttctttt ggccgcccct 541 tccaccttaa cttctgtgat tttctctaat cttcattttc ctcttagatc ttttctcttt 601 cttagcacct agcccccttc aagctctatc ataattcttt ctggcaactc ttggcctcaa 661 ttgtagtcct accccatgga atgcctcatt aggacccctt ccctgtcccc ccatatcaca 721 gccttccaaa caccctcaga agtaatcata cttcctgacc tcccatctcc agtgccgttt 781 cgaagcctgt ccctcagtcc cctttgacca gtaatctctt cttccttgct tttcattcca 841 aaaatgcttc aggccaatac tggcaagttc tagggggccc agtgtctggg ctgagcattg 901 ggacaggcag ggcaatgctg ggcacacaca ccatggaagt gactgtctac catcgccggg 961 gatcccggag ctatgtgcct cttgctcatt ccagctcagc cttcaccatt actggtaagg 1021 gttcaggaag ggcaaggcca gttgtagggc aaagagaagg cagggaggct tggatggact 1081 gcaaaggaga aaggtgaaat gctgtgcaaa cttaaagtag aagggccagg aagacctagg 1141 cagagaaatg tgaggcttag tgccagtgaa gggccagcca gtcagcttgg agttggaggg 1201 tgtggctgtg aaaggagaag ctgtggctca ggcctggttc tcaccttttc tggctccaat 1261 cccagaccag gtgcctttct ccgtgagcgt gtcccagttg cgggccttgg atggagggaa 1321 caagcacttc ctgagaaatc agcctctgac ctttgccctc cagctccatg accccagtgg 1381 ctatctggct gaagctgacc tctcctacac ctgggacttt ggagacagta gtggaaccct 1441 gatctctcgg gcacctgtgg tcactcatac ttacctggag cctggcccag tcactgccca 1501 ggtggtcctg caggctgcca ttcctctcac ctcctgtggc tcctccccag ttccaggcac 1561 cacagatggg cacaggccaa ctgcagaggc ccctaacacc acagctggcc aagtgcctac 1621 tacagaagtt gtgggtacta cacctggtca ggcgccaact gcagagccct ctggaaccac 1681 atctgtgcag gtgccaacca ctgaagtcat aagcactgca cctgtgcaga tgccaactgc 1741 agagagcaca ggtatgacac ctgagaaggt gccagtttca gaggtcatgg gtaccacact 1801 ggcagagatg tcaactccag aggctacagg tatgacacct gcagaggtat caattgtggt 1861 gctttctgga accacagctg cacaggtaac aactacagag tgggtggaga ccacagctag 1921 agagctacct atccctgagc ctgaaggtcc agatgccagc tcaatcatgt ctacggaaag 1981 tattacaggt tccctgggcc ccctgctgga tggtacagcc accttaaggc tggtgaagag 2041 acaagtcccc ctggattgtg ttctgtatcg atatggttcc ttttccgtca ccctggacat 2101 tgtccagggt attgaaagtg ccgagatcct gcaggctgtg ccgtccggtg agggggatgc 2161 atttgagctg actgtgtcct gccaaggcgg gctgcccaag gaagcctgca tggagatctc 2221 atcgccaggg tgccagcccc ctgcccagcg gctgtgccag cctgtgctac ccagcccagc 2281 ctgccagctg gttctgcacc agatactgaa gggtggctcg gggacatact gcctcaatgt 2341 gtctctggct gataccaaca gcctggcagt ggtcagcacc cagcttatca tgcctggtag 2401 gtccttggac agagactaag tgaggaggga agtggataga ggggacagct ggcaagcagc 2461 agacatgagt gaagcagtgc ctgggattct tctcacaggt caagaagcag gccttgggca 2521 ggttccgctg atcgtgggca tcttgctggt gttgatggct gtggtccttg catctctgat 2581 atataggcgc agacttatga agcaagactt ctccgtaccc cagttgccac atagcagcag 2641 tcactggctg cgtctacccc gcatcttctg ctcttgtccc attggtgaga atagccccct 2701 cctcagtggg cagcaggtct gagtactctc atatgatgct gtgattttcc tggagttgac 2761 agaaacacct atatttcccc cagtcttccc tgggagacta ctattaactg aaataaa // Homo sapiens kallikrein 3, (prostate specific antigen) (KLK3), mRNA. ACCESSION NM_001648 VERSTON NM_001648.1 GI:4502172 SEQ ID NO 78 /translation = “MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVAS RGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNR FLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCV DLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPER PSLYTKVVHYRKWIKDTIVANP” SEQ ID NO 86 ORIGIN 1 agccccaagc ttaccacctg cacccggaga gctgtgtgtc accatgtggg tcccggttgt 61 cttcctcacc ctgtccgtga cgtggattgg tgctgcaccc ctcatcctgt ctcggattgt 121 gggaggctgg gagtgcgaga agcattccca accctggcag gtgcttgtgg cctctcgtgg 181 cagggcagtc tgcggcggtg ttctggtgca cccccagtgg gtcctcacag ctgcccactg 241 catcaggaac aaaagcgtga tcttgctggg tcggcacagc ctgtttcatc ctgaagacac 301 aggccaggta tttcaggtca gccacagctt cccacacccg ctctacgata tgagcctcct 361 gaagaatcga ttcctcaggc caggtgatga ctccagccac gacctcatgc tgctccgcct 421 gtcagagcct gccgagctca cggatgctgt gaaggtcatg gacctgccca cccaggagcc 481 agcactgggg accacctgct acgcctcagg ctggggcagc attgaaccag aggagttctt 541 gaccccaaag aaacttcagt gtgtggacct ccatgttatt tccaatgacg tgtgtgcgca 601 agttcaccct cagaaggtga ccaagttcat gctgtgtgct ggacgctgga cagggggcaa 661 aagcacctgc tcgggtgatt ctgggggccc acttgtctgt aatggtgtgc ttcaaggtat 721 cacgtcatgg ggcagtgaac catgtgccct gcccgaaagg ccttccctgt acaccaaggt 781 ggtgcattac cggaagtgga tcaaggacac catcgtggcc aacccctgag cacccctatc 841 aaccccctat tgtagtaaac ttggaacctt ggaaatgacc aggccaagac tcaagcctcc 901 ccagttctac tgacctttgt ccttaggtgt gaggtccagg gttgctagga aaagaaatca 961 gcagacacag gtgtagacca gagtgtttct taaatggtgt aattttgtcc tctctgtgtc 1021 ctggggaata ctggccatgc ctggagacat atcactcaat ttctctgagg acacagatag 1081 gatggggtgt ctgtgttatt tgtggggtac agagatgaaa gaggggtggg atccacactg 1141 agagagtgga gagtgacatg tgctggacac tgtccatgaa gcactgagca gaagctggag 1201 gcacaacgca ccagacactc acagcaagga tggagctgaa aacataaccc actctgtcct 1261 ggaggcactg ggaagcctag agaaggctgt gagccaagga gggagggtct tcctttggca 1321 tgggatgggg atgaagtaag gagagggact ggaccccctg gaagctgatt cactatgggg 1381 ggaggtgtat tgaagtcctc cagacaaccc tcagatttga tgatttccta gtagaactca 1441 cagaaataaa gagctgttat actgtg // Human autoimmunogenic cancer/testis antigen NY-ESO-1 mRNA, complete cds. ACCESSION U87459 VERSION U87459.1 GI:1890098 SEQ ID NO 74 /translation=“MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEA GATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLL EFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTA ADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR” SEQ ID NO 84 ORIGIN 1 atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccgga gccatgcagg 61 ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccagga ggccctggca 121 ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc acgggcggca 181 gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gccccgcggg 241 gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gccagggggc 301 cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc atggaagcag 361 agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca ggggtgcttc 421 tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gcagaccacc 481 gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg atgtggatca 541 cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cgctaagccc 601 agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtccc agcacgagtg 661 gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggctt acatgtttgt 721 ttctgtagaa aataaaactg agctacgaaa aa // LAGE-1a protein [Homo sapiens]. ACCESSION CAA11116 PID g3255959 VERSION CAA11116.1 GI:3255959 SEQ ID NO 75 ORIGIN 1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgprgga 61 prgphggaas aqdgrcpcga rrpdsrllel hitmpfsspm eaelvrrils rdaaplprpg 121 avlkdftvsg nllfirltaa dhrqlqlsis sclqqlsllm witqcflpvf laqapsgqrr 181 // LACE-1b protein [Homo sapiens]. ACCESSION CAA11117 PID g3255960 VERSION CAA11117.1 GI:3255960 SEQ ID NO 76 ORIGIN 1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgprgga 61 prgphggaas aqdgrcpcga rrpdsrllel hitmpfsspm eaelvrrils rdaaplprpg 121 avlkciftvsg nllfmsvwdq dregagrmrv vgwglgsasp egqkardlrt pkhkvseqrp 181 gtpgppppeg aqgdgcrgva fnvmfsaphi // Human antigen (MACE-1) gene, complete cds. ACCESSION M77481 VERSION M77481.1 GI:416114 SEQ ID NO 71 /translation=“MSLEQRSLHCKPEEALEAQQEALGLVCVQAATSSSSP LVLGTLEEVPTAGSTDPPQSPQGASAFPTTINFTRQRQPSEGSSSREEEGP STSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHC FPEIFGKASESLQLVFGIDVKEADPTGHSYVLVTCLGLSYDGLLGDNQIMP KTGFLIIVLVMIAMEGGHAPEEEIWEELSVMEVYDGREHSAYGEPRKLLTQ DLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVRF FFPSLREAALREEEEGV” SEQ ID NO 81 ORIGIN 1 ggatccaggc cctgccagga aaaatataag ggccctgcgt gagaacagag ggggtcatcc 61 actgcatgag agtggggatg tcacagagtc cagcccaccc tcctggtagc actgagaagc 121 cagggctgtg cttgcggtct gcaccctgag ggcccgtgga ttcctcttcc tggagctcca 181 ggaaccaggc agtgaggcct tggtctgaga cagtatcctc aggtcacaga gcagaggatg 241 cacagggtgt gccagcagtg aatgtttgcc ctgaatgcac accaagggcc ccacctgcca 301 caggacacat aggactccac agagtctggc ctcacctccc tactgtcagt cctgtagaat 361 cgacctctgc tggccggctg taccctgagt accctctcac ttcctccttc aggttttcag 421 gggacaggcc aacccagagg acaggattcc ctggaggcca cagaggagca ccaaggagaa 481 gatctgtaag taggcctttg ttagagtctc caaggttcag ttctcagctg aggcctctca 541 cacactccct ctctccccag gcctgtgggt cttcattgcc cagctcctgc ccacactcct 601 gcctgctgcc ctgacgagag tcatcatgtc tcttgagcag aggagtctgc actgcaagcc 661 tgaggaagcc cttgaggccc aacaagaggc cctgggcctg gtgtgtgtgc aggctgccac 721 ctcctcctcc tctcctctgg tcctgggcac cctggaggag gtgcccactg ctgggtcaac 781 agatcctccc cagagtcctc agggagcctc cgcctttccc actaccatca acttcactcg 841 acagaggcaa cccagtgagg gttccagcag ccgtgaagag gaggggccaa gcacctcttg 901 tatcctggag tccttgttcc gagcagtaat cactaagaag gtggctgatt tggttggttt 961 tctgctcctc aaatatcgag ccagggagcc agtcacaaag gcagaaatgc tggagagtgt 1021 catcaaaaat tacaagcact gttttcctga gatcttcggc aaagcctctg agtccttgca 1081 gctggtcttt ggcattgacg tgaaggaagc agaccccacc ggccactcct atgtccttgt 1141 cacctgccta ggtctctcct atgatggcct gctgggtgat aatcagatca tgcccaagac 1201 aggcttcctg ataattgtcc tggtcatgat tgcaatggag ggcggccatg ctcctgagga 1261 ggaaatctgg gaggagctga gtgtgatgga ggtgtatgat gggagggagc acagtgccta 1321 tggggagccc aggaagctgc tcacccaaga tttggtgcag gaaaagtacc tggagtaccg 1381 gcaggtgccg gacagtgatc ccgcacgcta tgagttcctg tggggtccaa gggccctcgc 1441 tgaaaccagc tatgtgaaag tccttgagta tgtgatcaag gtcagtgcaa gagttcgctt 1501 tttcttccca tccctgcgtg aagcagcttt gagagaggag gaagagggag tctgagcatg 1561 agttgcagcc aaggccagtg ggagggggac tgggccagtg caccttccag ggccgcgtcc 1621 agcagcttcc cctgcctcgt gtgacatgag gcccattctt cactctgaag agagcggtca 1681 gtgttctcag tagtaggttt ctgttctatt gggtgacttg gagatttatc tttgttctct 1741 tttggaattg ttcaaatgtt tttttttaag ggatggttga atgaacttca gcatccaagt 1801 ttatgaatga cagcagtcac acagttctgt gtatatagtt taagggtaag agtcttgtgt 1861 tttattcaga ttgggaaatc cattctattt tgtgaattgg gataataaca gcagtggaat 1921 aagtacttag aaatgtgaaa aatgagcagt aaaatagatg agataaagaa ctaaagaaat 1981 taagagatag tcaattcttg ccttatacct cagtctattc tgtaaaattt ttaaagatat 2041 atgcatacct ggatttcctt ggcttctttg agaatgtaag agaaattaaa tctgaataaa 2101 gaattcttcc tgttcactgg ctcttttctt ctccatgcac tgagcatctg ctttttggaa 2161 ggccctgggt tagtagtgga gatgctaagg taagccagac tcatacccac ccatagggtc 2221 gtagagtcta ggagctgcag tcacgtaatc gaggtggcaa gatgtcctct aaagatgtag 2281 ggaaaagtga gagaggggtg agggtgtggg gctccgggtg agagtggtgg agtgtcaatg 2341 ccctgagctg gggcattttg ggctttggga aactgcagtt ccttctgggg gagctgattg 2401 taatgatctt gggtggatcc // Human MAGE-2 gene exons 1-4, complete cds. ACCESSION L18920 VERSION L18920.1 GI:436180 SEQ ID NO 72 /translation = “MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQQTASSSSTLVEV TLGEVPAADSPSPPHSPQGASSFSTTINYTLWRQSDEGSSNQEEEGPRMFPDLE SEFQAAISRKMVELVHFLLLKYRAREPVTKAEMLESVLRNCQDFFPVIFSKASEYLQLVFGI EVVEVVPISHLYILVTCLGLSYDGLLGDNQVMPKTGLLIIVLAIIAIEGDCAPEEKIWEELS MLEVFEGREDSVFAHPRKLLMQDLVQENYLEYRQVPGSDPACYEFLWGPRALIETSYVKVLH HTLKIGGEPHISYPPLHERALREGEE” SEQ ID NO 82 ORIGIN 1 attccttcat caaacagcca ggagtgagga agaggaccct cctgagtgag gactgaggat 61 ccaccctcac cacatagtgg gaccacagaa tccagctcag cccctcttgt cagccctggt 121 acacactggc aatgatctca ccccgagcac acccctcccc ccaatgccac ttcgggccga 181 ctcagagtca gagacttggt ctgaggggag cagacacaat cggcagagga tggcggtcca 241 ggctcagtct ggcatccaag tcaggacctt gagggatgac caaaggcccc tcccaccccc 301 aactcccccg acaccaccag gatctacagc ctcaggatcc ccgtcccaat ccctacccct 361 acaccaacac catcttcatg cttaccccca cccccccatc cagatcccca tccgggcaga 421 atccggttcc acccttgccg tgaacccagg gaagtcacgg gcccggatgt gacgccactg 481 acttgcacat tggaggtcag aggacagcga gattctcgcc ctgagcaacg gcctgacgtc 541 ggcggaggga agcaggcgca ggctccgtga ggaggcaagg taagacgccg agggaggact 601 gaggcgggcc tcaccccaga cagagggccc ccaataatcc agcgctgcct ctgctgccgg 661 gcctggacca ccctgcaggg gaagacttct caggctcagt cgccaccacc tcaccccgcc 721 accccccgcc gctttaaccg cagggaactc tggcgtaaga gctttgtgtg accagggcag 781 ggctggttag aagtgctcag ggcccagact cagccaggaa tcaaggtcag gaccccaaga 841 ggggactgag ggcaacccac cccctaccct cactaccaat cccatccccc aacaccaacc 901 ccacccccat ccctcaaaca ccaaccccac ccccaaaccc cattcccatc tcctccccca 961 ccaccatcct ggcagaatcc ggctttgccc ctgcaatcaa cccacggaag ctccgggaat 1021 ggcggccaag cacgcggatc ctgacgttca catgtacggc taagggaggg aaggggttgg 1081 gtctcgtgag tatggccttt gggatgcaga ggaagggccc aggcctcctg gaagacagtg 1141 gagtccttag gggacccagc atgccaggac agggggccca ctgtacccct gtctcaaact 1201 gagccacctt ttcattcagc cgagggaatc ctagggatgc agacccactt cagcaggggg 1261 ttggggccca gcctgcgagg agtcaagggg aggaagaaga gggaggactg aggggacctt 1321 ggagtccaga tcagtggcaa ccttgggctg ggggatcctg ggcacagtgg ccgaatgtgc 1381 cccgtgctca ttgcaccttc agggtgacag agagttgagg gctgtggtct gagggctggg 1441 acttcaggtc agcagaggga ggaatcccag gatctgccgg acccaaggtg tgcccccttc 1501 atgaggactg gggatacccc cggcccagaa agaagggatg ccacagagtc tggaagtccc 1561 ttgttcttag ctctggggga acctgatcag ggatggccct aagtgacaat ctcatttgta 1621 ccacaggcag gaggttgggg aaccctcagg gagataaggt gttggtgtaa agaggagctg 1681 tctgctcatt tcagggggtt gggggttgag aaagggcagt ccctggcagg agtaaagatg 1741 agtaacccac aggaggccat cataacgttc accctagaac caaaggggtc agccctggac 1801 aacgcacgtg ggggtaacag gatgtggccc ctcctcactt gtctttccag atctcaggga 1861 gttgatgacc ttgttttcag aaggtgactc aggtcaacac aggggcccca tctggtcgac 1921 agatgcagtg gttctaggat ctgccaagca tccaggtgga gagcctgagg taggattgag 1981 ggtacccctg ggccagaatg cagcaagggg gccccataga aatctgccct gcccctgcgg 2041 ttacttcaga gaccctgggc agggctgtca gctgaagtcc ctccattatc ctgggatctt 2101 tgatgtcagg gaaggggagg ccttggtctg aaggggctgg agtcaggtca gtagagggag 2161 ggtctcaggc cctgccagga gtggacgtga ggaccaagcg gactcgtcac ccaggacaca 2221 tggactccaa tgaatttgga catctctcgt tgtccttcgc gggaggacct ggtcacgtat 2281 ggccagatgt gggtcccctc atatccttct gtaccatatc agggatgtga gttcttgaca 2341 tgagagattc tcaagccagc aaaagggtgg gattaggccc tacaaggaga aaggtgaggg 2401 ccctgagtga gcacagaggg gaccctccac ccaagtagag tggggacctc acggagtctg 2461 gccaaccctg ctgagacttc tgggaatccg tggctgtgct tgcagtctgc acactgaagg 2521 cccgtgcatt cctctcccag gaatcaggag ctccaggaac caggcagtga ggccttggtc 2581 tgagtcagtg tcctcaggtc acagagcaga ggggacgcag acagtgccaa cactgaaggt 2641 ttgcctggaa tgcacaccaa gggccccacc cgcccagaac aaatgggact ccagagggcc 2701 tggcctcacc ctccctattc tcagtcctgc agcctgagca tgtgctggcc ggctgtaccc 2761 tgaggtgccc tcccacttcc tccttcaggt tctgaggggg acaggctgac aagtaggacc 2821 cgaggcactg gaggagcatt gaaggagaag atctgtaagt aagcctttgt cagagcctcc 2881 aaggttcagt tcagttctca cctaaggcct cacacacgct ccttctctcc ccaggcctgt 2941 gggtcttcat tgcccagctc ctgcccgcac tcctgcctgc tgccctgacc agagtcatca 3001 tgcctcttga gcagaggagt cagcactgca agcctgaaga aggccttgag gcccgaggag 3061 aggccctggg cctggtgggt gcgcaggctc ctgctactga ggagcagcag accgcttctt 3121 cctcttctac tctagtggaa gttaccctgg gggaggtgcc tgctgccgac tcaccgagtc 3181 ctccccacag tcctcaggga gcctccagct tctcgactac catcaactac actctttgga 3241 gacaatccga tgagggctcc agcaaccaag aagaggaggg gccaagaatg tttcccgacc 3301 tggagtccga gttccaagca gcaatcagta ggaagatggt tgagttggtt cattttctgc 3361 tcctcaagta tcgagccagg gagccggtca caaaggcaga aatgctggag agtgtcctca 3421 gaaattgcca ggacttcttt cccgtgatct tcagcaaagc ctccgagtac ttgcagctgg 3481 tctttggcat cgaggtggtg gaagtggtcc ccatcagcca cttgtacatc cttgtcacct 3541 gcctgggcct ctcctacgat ggcctgctgg gcgacaatca ggtcatgccc aagacaggcc 3601 tcctgataat cgtcctggcc ataatcgcaa tagagggcga ctgtgcccct gaggagaaaa 3661 tctgggagga gctgagtatg ttggaggtgt ttgaggggag ggaggacagt gtcttcgcac 3721 atcccaggaa gctgctcatg caagatctgg tgcaggaaaa ctacctggag taccggcagg 3781 tgcccggcag tgatcctgca tgctacgagt tcctgtgggg tccaagggcc ctcattgaaa 3841 ccagctatgt gaaagtcctg caccatacac taaagatcgg tggagaacct cacatttcct 3901 acccacccct gcatgaacgg gctttgagag agggagaaga gtgagtctca gcacatgttg 3961 cagccagggc cagtgggagg gggtctgggc cagtgcacct tccagggccc catccattag 4021 cttccactgc ctcgtgtgat atgaggccca ttcctgcctc tttgaagaga gcagtcagca 4081 ttcttagcag tgagtttctg ttctgttgga tgactttgag atttatcttt ctttcctgtt 4141 ggaattgttc aaatgttcct tttaacaaat ggttggatga acttcagcat ccaagtttat 4201 gaatgacagt agtcacacat agtgctgttt atatagttta ggggtaagag tcctgttttt 4261 tattcagatt gggaaatcca ttccattttg tgagttgtca cataataaca gcagtggaat 4321 atgtatttgc ctatattgtg aacgaattag cagtaaaata catgatacaa ggaactcaaa 4381 agatagttaa ttcttgcctt atacctcagt ctattatgta aaattaaaaa tatgtgtatg 4441 tttttgcttc tttgagaatg caaaagaaat taaatctgaa taaattcttc ctgttcactg 4501 gctcatttct ttaccattca ctcagcatct gctctgtgga aggccctggt agtagtggg // Human MAGE-3 antigen (MAGE-3) gene, complete cds. ACCESSION U03735 VERSION U03735.1 GI:468825 SEQ ID NO 73 /translation = “MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLG EVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAEL VHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATC LGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLL TQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLRE GEE” SEQ ID NO 83 ORIGIN 1 acgcaggcag tgatgtcacc cagaccacac cccttccccc aatgccactt cagggggtac 61 tcagagtcag agacttggtc tgaggggagc agaagcaatc tgcagaggat ggcggtccag 121 gctcagccag gcatcaactt caggaccctg agggatgacc gaaggccccg cccacccacc 181 cccaactccc ccgaccccac caggatctac agcctcagga cccccgtccc aatccttacc 241 ccttgcccca tcaccatctt catgcttacc tccaccccca tccgatcccc atccaggcag 301 aatccagttc cacccctgcc cggaacccag ggtagtaccg ttgccaggat gtgacgccac 361 tgacttgcgc attggaggtc agaagaccgc gagattctcg ccctgagcaa cgagcgacgg 421 cctgacgtcg gcggagggaa gccggcccag gctcggtgag gaggcaaggt aagacgctga 481 gggaggactg aggcgggcct cacctcagac agagggcctc aaataatcca gtgctgcctc 541 tgctgccggg cctgggccac cccgcagggg aagacttcca ggctgggtcg ccactacctc 601 accccgccga cccccgccgc tttagccacg gggaactctg gggacagagc ttaatgtggc 661 cagggcaggg ctggttagaa gaggtcaggg cccacgctgt ggcaggaatc aaggtcagga 721 ccccgagagg gaactgaggg cagcctaacc accaccctca ccaccattcc cgtcccccaa 781 cacccaaccc cacccccatc ccccattccc atccccaccc ccaccectat cctggcagaa 841 tccgggcttt gcccctggta tcaagtcacg gaagctccgg gaatggcggc caggcacgtg 901 agtcctgagg ttcacatcta cggctaaggg agggaagggg ttcggtatcg cgagtatggc 961 cgttgggagg cagcgaaagg gcccaggcct cctggaagac agtggagtcc tgaggggacc 1021 cagcatgcca ggacaggggg cccactgtac ccctgtctca aaccgaggca ccttttcatt 1081 cggctacggg aatcctaggg atgcagaccc acttcagcag ggggttgggg cccagccctg 1141 cgaggagtca tggggaggaa gaagagggag gactgagggg accttggagt ccagatcagt 1201 ggcaaccttg ggctggggga tgctgggcac agtggccaaa tgtgctctgt gctcattgcg 1261 ccttcagggt gaccagagag ttgagggctg tggtctgaag agtgggactt caggtcagca 1321 gagggaggaa tcccaggatc tgcagggccc aaggtgtacc cccaaggggc ccctatgtgg 1381 tggacagatg cagtggtcct aggatctgcc aagcatccag gtgaagagac tgagggagga 1441 ttgagggtac ccctgggaca gaatgcggac tgggggcccc ataaaaatct gccctgctcc 1501 tgctgttacc tcagagagcc tgggcagggc tgtcagctga ggtccctcca ttatcctagg 1561 atcactgatg tcagggaagg ggaagccttg gtctgagggg gctgcactca gggcagtaga 1621 gggaggctct cagaccctac taggagtgga ggtgaggacc aagcagtctc ctcacccagg 1681 gtacatggac ttcaataaat ttggacatct ctcgttgtcc tttccgggag gacctgggaa 1741 tgtatggcca gatgtgggtc ccctcatgtt tttctgtacc atatcaggta tgtgagttct 1801 tgacatgaga gattctcagg ccagcagaag ggagggatta ggccctataa ggagaaaggt 1861 gagggccctg agtgagcaca gaggggatcc tccaccccag tagagtgggg acctcacaga 1921 gtctggccaa ccctcctgac agttctggga atccgtggct gcgtttgctg tctgcacatt 1981 gggggcccgt ggattcctct cccaggaatc aggagctcca ggaacaaggc agtgaggact 2041 tggtctgagg cagtgtcctc aggtcacaga gtagaggggg ctcagatagt gccaacggtg 2101 aaggtttgcc ttggattcaa accaagggcc ccacctgccc cagaacacat ggactccaga 2161 gcgcctggcc tcaccctcaa tactttcagt cctgcagcct cagcatgcgc tggccggatg 2221 taccctgagg tgccctctca cttcctcctt caggttctga ggggacaggc tgacctggag 2281 gaccagaggc ccccggagga gcactgaagg agaagatctg taagtaagcc tttgttagag 2341 cctccaaggt tccattcagt actcagctga ggtctctcac atgctccctc tctccccagg 2401 ccagtgggtc tccattgccc agctcctgcc cacactcccg cctgttgccc tgaccagagt 2461 catcatgcct cttgagcaga ggagtcagca ctgcaagcct gaagaaggcc ttgaggcccg 2521 aggagaggcc ctgggcctgg tgggtgcgca ggctcctgct actgaggagc aggaggctgc 2581 ctcctcctct tctactctag ttgaagtcac cctgggggag gtgcctgctg ccgagtcacc 2641 agatcctccc cagagtcctc agggagcctc cagcctcccc actaccatga actaccctct 2701 ctggagccaa tcctatgagg actccagcaa ccaagaagag gaggggccaa gcaccttccc 2761 tgacctggag tccgagttcc aagcagcact cagtaggaag gtggccgagt tggttcattt 2821 tctgctcctc aagtatcgag ccagggagcc ggtcacaaag gcagaaatgc tggggagtgt 2881 cgtcggaaat tggcagtatt tctttcctgt gatcttcagc aaagcttcca gttccttgca 2941 gctggtcttt ggcatcgagc tgatggaagt ggaccccatc ggccacttgt acatctttgc 3001 cacctgcctg ggcctctcct acgatggcct gctgggtgac aatcagatca tgcccaaggc 3061 aggcctcctg ataatcgtcc tggccataat cgcaagagag ggcgactgtg cccctgagga 3121 gaaaatctgg gaggagctga gtgtgttaga ggtgtttgag gggagggaag acagtatctt 3181 gggggatccc aagaagctgc tcacccaaca tttcgtgcag gaaaactacc tggagtaccg 3241 gcaggtcccc ggcagtgatc ctgcatgtta tgaattcctg tggggtccaa gggccctcgt 3301 tgaaaccagc tatgtgaaag tcctgcacca tatggtaaag atcagtggag gacctcacat 3361 ttcctaccca cccctgcatg agtgggtttt gagagagggg gaagagtgag tctgagcacg 3421 agttgcagcc agggccagtg ggagggggtc tgggccagtg caccttccgg ggccgcatcc 3481 cttagtttcc actgcctcct gtgacgtgag gcccattctt cactctttga agcgagcagt 3541 cagcattctt agtagtgggt ttctgttctg ttggatgact ttgagattat tctttgtttc 3601 ctgttggagt tgttcaaatg ttccttttaa cggatggttg aatgagcgtc agcatccagg 3661 tttatgaatg acagtagtca cacatagtgc tgtttatata gtttaggagt aagagtcttg 3721 ttttttactc aaattgggaa atccattcca ttttgtgaat tgtgacataa taatagcagt 3781 ggtaaaagta tttgcttaaa attgtgagcg aattagcaat aacatacatg agataactca 3841 agaaatcaaa agatagttga ttcttgcctt gtacctcaat ctattctgta aaattaaaca 3901 aatatgcaaa ccaggatttc cttgacttct ttgagaatgc aagcgaaatt aaatctgaat 3961 aaataattct tcctcttcac tggctcgttt cttttccgtt cactcagcat ctgctctgtg 4021 ggaggccctg ggttagtagt ggggatgcta aggtaagcca gactcacgcc tacccatagg 4081 gctgtagagc ctaggacctg cagtcatata attaaggtgg tgagaagtcc tgtaagatgt 4141 agaggaaatg taagagaggg gtgagggtgt ggcgctccgg gtgagagtag tggagtgtca 4201 gtgc // Homo sapiens prostate stem cell antigen (PSCA) mRNA, complete cds. ACCESSION AF043498 VERSION AF043498.1 GI:2909843 SEQ ID NO 79 /translation = “MKAVLLALLMAGLALQPCTALLCYSCKAQVSNEDCLQVENCTQLGEQC WTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALL PALGLLLWGPGQL” SEQ ID NO 87 ORIGIN 1 agggagaggc agtgaccatg aaggctgtgc tgcttgccct gttgatggca ggcttggccc 61 tgcagccagg cactgccctg ctgtgctact cctgcaaagc ccaggtgagc aacgaggact 121 gcctgcaggt ggagaactgc acccagctgg gggagcagtg ctggaccgcg cgcatccgcg 181 cagttggcct cctgaccgtc atcagcaaag gctgcagctt gaactgcgtg gatgactcac 241 aggactacta cgtgggcaag aagaacatca cgtgctgtga caccgacttg tgcaacgcca 301 gcggggccca tgccctgcag ccggctgccg ccatccttgc gctgctccct gcactcggcc 361 tgctgctctg gggacccggc cagctatagg ctctgggggg ccccgctgca gcccacactg 421 ggtgtggtgc cccaggcctt tgtgccactc ctcacagaac ctggcccagt gggagcctgt 481 cctggttcct gaggcacatc ctaacgcaag tttgaccatg tatgtttgca ccccttttcc 541 ccnaaccctg accttcccat gggccttttc caggattccn accnggcaga tcagttttag 601 tganacanat ccgcntgcag atggcccctc caaccntttn tgttgntgtt tccatggccc 661 agcattttcc acccttaacc ctgtgttcag gcacttnttc ccccaggaag ccttccctgc 721 ccaccccatt tatgaattga gccaggtttg gtccgtggtg tcccccgcac ccagcagggg 781 acaggcaatc aggagggccc agtaaaggct gagatgaagt ggactgagta gaactggagg 841 acaagagttg acgtgagttc ctgggagttt ccagagatgg ggcctggagg cctggaggaa 901 ggggccaggc ctcacatttg tggggntccc gaatggcagc ctgagcacag cgtaggccct 961 taataaacac ctgttggata agccaaaaaa // GLANDULAR KALLIKREIN 1 PRECURSOR (TISSUE KALLIKREIN) (KIDNEY/PANCREAS/SALIVARY GLAND KALLIKREIN). ACCESSION P06870 PID g125170 VERSION P06870 GI:125170 SEQ ID NO 600 ORIGIN 1 mwflvlclal slggtgaapp iqsrivggwe ceqhsqpwqa alyhfstfqc ggilvhrqwv 61 ltaahcisdn yqlwlgrhnl fddentaqfv hvsesfphpg fnmsllenht rqadedyshd 121 lmllrltepa dtitdavkvv elptqepevg stclasgwgs iepenfsfpd dlqcvdlkil 181 pndecekahv qkvtdfmlcv ghleggkdtc vgdsggplmc dgvlqgvtsw gyvpcgtpnk 241 psvavrvlsy vkwiedtiae ns // ELASTASE 2A PRECURSOR. ACCESSION P08217 PID g119255 VERSION P08217 GI:119255 SEQ ID NO 601 ORIGIN 1 mirtlllstl vagalscgdp typpyvtrvv ggeearpnsw pwqvslqyss ngkwyhtcgg 61 slianswvlt aahcisssrt yrvglgrhnl yvaesgslav svskivvhkd wnsnqiskgn 121 diallklanp vsltdkiqla clppagtilp nnypcyvtgw grlqtngavp dvlqqgrllv 181 vdyatcsssa wwgssvktsm icaggdgvis scngdsggpl ncqasdgrwq vhgivsfgsr 241 lgcnyyhkps vftrvsnyid winsviann // pancreatic elastase IIB [Homo sapiens]. ACCESSION NP_056933 PID g7705648 VERSION NP_056933.1 GI:7705648 SEQ ID NO 602 ORIGIN 1 mirtlllstl vagalscgvs tyapdmsrml ggeearpnsw pwqvslqyss ngqwyhtcgg 61 slianswvlt aahcisssri yrvmlgqhnl yvaesgslav svskivvhkd wnsnqvskgn 121 diallklanp vsltdkiqla clppagtilp nnypcyvtgw grlqtngalp ddlkqgrllv 181 vdyatcsssg wwgstvktnm icaggdgvic tcngdsggpl ncqasdgrwe vhgigsltsv 241 lgcnyyykps iftrvsnynd winsviann // PRAME Homo sapiens preferentially expressed antigen in melanoma (PRAME), mRNA. ACCESSION NM_006115 VERSION NM_006115.1 GI:5174640 SEQ ID NO 77 /translation = “MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRE LFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRR WKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDL FLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKL PTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRG RLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERA SATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLY PVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCF MPN” SEQ ID NO 85 ORIGIN 1 gcttcagggt acagctcccc cgcagccaga agccgggcct gcagcccctc agcaccgctc 61 cgggacaccc cacccgcttc ccaggcgtga cctgtcaaca gcaacttcgc ggtgtggtga 121 actctctgag gaaaaaccat tttgattatt actctcagac gtgcgtggca acaagtgact 181 gagacctaga aatccaagcg ttggaggtcc tgaggccagc ctaagtcgct tcaaaatgga 241 acgaaggcgt ttgtggggtt ccattcagag ccgatacatc agcatgagtg tgtggacaag 301 cccacggaga cttgtggagc tggcagggca gagcctgctg aaggatgagg ccctggccat 361 tgccgccctg gagttgctgc ccagggagct cttcccgcca ctcttcatgg cagcctttga 421 cgggagacac agccagaccc tgaaggcaat ggtgcaggcc tggcccttca cctgcctccc 481 tctgggagtg ctgatgaagg gacaacatct tcacctggag accttcaaag ctgtgcttga 541 tggacttgat gtgctccttg cccaggaggt tcgccccagg aggtggaaac ttcaagtgct 601 ggatttacgg aagaactctc atcaggactt ctggactgta tggtctggaa acagggccag 661 tctgtactca tttccagagc cagaagcagc tcagcccatg acaaagaagc gaaaagtaga 721 tggtttgagc acagaggcag agcagccctt cattccagta gaggtgctcg tagacctgtt 781 cctcaaggaa ggtgcctgtg atgaattgtt ctcctacctc attgagaaag tgaagcgaaa 841 gaaaaatgta ctacgcctgt gctgtaagaa gctgaagatt tttgcaatgc ccatgcagga 901 tatcaagatg atcctgaaaa tggtgcagct ggactctatt gaagatttgg aagtgacttg 961 tacctggaag ctacccacct tggcgaaatt ttctccttac ctgggccaga tgattaatct 1021 gcgtagactc ctcctctccc acatocatgo atcttcctac atttccccgg agaaggaaga 1081 gcagtatatc gcccagttca cctctcagtt cctcagtctg cagtgcctgc aggctctcta 1141 tgtggactct ttatttttcc ttagaggccg cctggatcag ttgctcaggc acgtgatgaa 1201 ccccttggaa accctctcaa taactaactg ccggctttcg gaaggggatg tgatgcatct 1261 gtcccagagt cccagcgtca gtcagctaag tgtcctgagt ctaagtgggg tcatgctgac 1321 cgatgtaagt cccgagcccc tccaagctct gctggagaga gcctctgcca ccctccagga 1381 cctggtcttt gatgagtgtg ggatcacgga tgatcagctc cttgccctcc tgccttccct 1441 gagccactgc tcccagctta caaccttaag cttctacggg aattccatct ccatatctgc 1501 cttgcagagt ctcctgcagc acctcatcgg gctgagcaat ctgacccacg tgctgtatcc 1561 tgtccccctg gagagttatg aggacatcca tggtaccctc cacctggaga ggcttgccta 1621 tctgcatgcc aggctcaggg agttgctgtg tgagttgggg cggcccagca tggtctggct 1681 tagtgccaac ccctgtcctc actgtgggga cagaaccttc tatgacccgg agcccatcct 1741 gtgcccctgt ttcatgccta actagctggg tgcacatatc aaatgcttca ttctgcatac 1801 ttggacacta aagccaggat gtgcatgcat cttgaagcaa caaagcagcc acagtttcag 1861 acaaatgttc agtgtgagtg aggaaaacat gttcagtgag gaaaaaacat tcagacaaat 1921 gttcagtgag gaaaaaaagg ggaagttggg gataggcaga tgttgacttg aggagttaat 1981 gtgatctttg gggagataca tcttatagag ttagaaatag aatctgaatt tctaaaggga 2041 gattctggct tgggaagtac atgtaggagt taatccctgt gtagactgtt gtaaagaaac 2101 tgttgaaaat aaagagaagc aatgtgaagc aaaaaaaaaa aaaaaaaa ED-B domain of Fibronectin Human fibronectin gene ED-B region. ACCESSION X07717 VERSION X07717.1 GI:31406 SEQ ID NO 590 /translation = “CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSF VDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID YDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTW” SEQ ID NO 591 ORIGIN 1 ctgcactttt gataacctga gtcccggcct ggagtacaat gtcagtgttt acactgtcaa 61 ggatgacaag gaaagtgtcc ctatctctga taccatcatc ccaggtaata gaaaataagc 121 tgctatcctg agagtgacat tccaataaga gtggggatta gcatcttaat ccccagatgc 181 ttaagggtgt caactatatt tgggatttaa ttccgatctc ccagctgcac tttccaaaac 241 caagaagtca aagcagcgat ttggacaaaa tgcttgctgt taacactgct ttactgtctg 301 tgcttcactg ggatgctgtg tgttgcagcg agtatgtaat ggagtggcag ccatggcttt 361 aactctgtat tgtctgctca catggaagta tgactaaaac actgtcacgt gtctgtactc 421 agtactgata ggctcaaagt aatatggtaa atgcatccca tcagtacatt tctgcccgat 481 tttacaatcc atatcaattt ccaacagctg cctatttcat cttgcagttt caaatccttc 541 tttttgaaaa ttggatttta aaaaaaagtt aagtaaaagt cacaccttca gggttgttct 601 ttcttgtggc cttgaaagac aacattgcaa aggcctgtcc taaggatagg cttgtttgtc 661 cattgggtta taacataatg aaagcattgg acagatcgtg tccccctttg gactcttcag 721 tagaatgctt ttactaacgc taattacatg ttttgattat gaatgaacct aaaatagtgg 781 caatggcctt aacctaggcc tgtctttcct cagcctgaat gtgcttttga atggcacatt 841 tcacaccata cattcataat gcattagcgt tatggccatg atgttgtcat gagttttgta 901 tgggagaaaa aaaatcaatt tatcacccat ttattatttt ttccggttgt tcatgcaagc 961 ttattttcta ctaaaacagt tttggaatta ttaaaagcat tgctgatact tacttcagat 1021 attatgtcta ggctctaaga atggtttcga catcctaaac agccatatga tttttaggaa 1081 tctgaacagt tcaaattgta ccctttaagg atgttttcaa aatgtaaaaa atatatatat 1141 atatatatat tccctaaaag aatattcctg tttattcttc tagggaagca aactgttcat 1201 gatgcttagg aagtcttttc agagaattta aaacagattg catattacca tcattgcttt 1261 aacattccac caattttact actagtaacc tgatatacac tgctttattt tttcctcttt 1321 ttttccctct attttccttt tgcctccccc tccctttgct ttgtaactca atagaggtgc 1381 cccaactcac tgacctaagc tttgttgata taaccgattc aagcatcggc ctgaggtgga 1441 ccccgctaaa ctcttccacc attattgggt accgcatcac agtagttgcg gcaggagaag 1501 gtatccctat ttttgaagat tttgtggact cctcagtagg atactacaca gtcacagggc 1561 tggagccggg cattgactat gatatcagcg ttatcactct cattaatggc ggcgagagtg 1621 cccctactac actgacacaa caaacgggtg aattttgaaa acttctgcgt ttgagacata 1681 gatggtgttg catgctgcca ccagttactc cggttaaata tggatgtttc atgggggaag 1741 tcagcaattg gccaaagatt cagataggtg gaattggggg gataaggaat caaatgcatc 1801 tgctaaactg attggagaaa aacacatgca atatcttcag tacactctca tttaaaccac 1861 aagtagatat aaagcctaga gaaatacaga tgtctgctct gttaaatata aaatagcaaa 1921 tgttcattca atttgaagac ctagaatttt tcttcttaaa taccaaacac gaataccaaa 1981 ttgcgtaagt accaattgat aagaatatat caccaaaatg taccatcatg ctcttccttc 2041 taccctttga taaactctac catgctcctt ctttgtagct aaaaacccat caaaatttag 2101 ggtagagtgg atgggcattg ttttgaggta ggagaaaagt aaacttggga ccattctagg 2161 ttttgttgct gtcactaggt aaagaaacac ctctttaacc acagtctggg gacaagcatg 2221 caacatttta aaggttctct gctgtgcatg ggaaaagaaa catgctgaga accaatttgc 2281 atgaacatgt tcacttgtaa gtagaattca ctgaatggaa ctgtagctct agatatctca 2341 catgggggga agtttaggac cctcttgtct ttttgtctgt gtgcatgtat ttctttgtaa 2401 agtactgcta tgtttctctt tgctgtgtgg caacttaagc ctcttcggcc tgggataaaa 2461 taatctgcag tggtattaat aatgtacata aagtcaacat atttgaaagt agattaaaat 2521 cttttttaaa tatatcaatg atggcaaaaa ggttaaaggg ggcctaacag tactgtgtgt 2581 agtgttttat ttttaacagt agtacactat aacttaaaat agacttagat tagactgttt 2641 gcatgattat gattctgttt cctttatgca tgaaatattg attttacctt tccagctact 2701 tcgttagctt taattttaaa atacattaac tgagtcttcc ttcttgttcg aaaccagctg 2761 ttcctcctcc cactgacctg cgattcacca acattggtcc agacaccatg cgtgtcacct 2821 ggg // CEA Homo sapiens carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mRNA. ACCESSION NM_004363 VERSION NM_004363.1 GI:11386170 SEQ ID NO 592 /translation = “MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFN VAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIY PNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDK DAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQ NPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGT FQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNP VEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYE CGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWL IDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSN NSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDA RAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQ YSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPG LSAGATVGIMIGVLVGVALI” SEQ ID NO 593 ORIGIN 1 ctcagggcag agggaggaag gacagcagac cagacagtca cagcagcctt gacaaaacgt 61 tcctggaact caagctcttc tccacagagg aggacagagc agacagcaga gaccatggag 121 tctccctcgg cccctcccca cagatggtgc atcccctggc agaggctcct gctcacagcc 181 tcacttctaa ccttctggaa cccgcccacc actgccaagc tcactattga atccacgccg 241 ttcaatgtcg cagaggggaa ggaggtgctt ctacttgtcc acaatctgcc ccagcatctt 301 tttggctaca gctggtacaa aggtgaaaga gtggatggca accgtcaaat tataggatat 361 gtaataggaa ctcaacaagc taccccaggg cccgcataca gtggtcgaga gataatatac 421 cccaatgcat ccctgctgat ccagaacatc atccagaatg acacaggatt ctacacccta 481 cacgtcataa agtcagatct tgtgaatgaa gaagcaactg gccagttccg ggtatacccg 541 gagatgocca agccctccat ctccagcaac aactccaaac ccgtggagga caaggatgct 601 gtggccttca cctgtgaacc tgagactcag gacgcaacct acctgtggtg ggtaaacaat 661 cagagcctcc cggtcagtcc caggctgcag ctgtccaatg gcaacaggac cctcactcta 721 ttcaatgtca caagaaatga cacagcaagc tacaaatgtg aaacccagaa cccagtgagt 781 gccaggcgca gtgattcagt catcctgaat gtcctctatg gcccggatgc ccccaccatt 841 tcccctctaa acacatctta cagatcaggg gaaaatctga acctctcctg ccacgcagcc 901 tctaacccac ctgcacagta ctcttggttt gtcaatggga ctttccagca atccacccaa 961 gagctcttta tccccaacat cactgtgaat aatagtggat cctatacgtg ccaagcccat 1021 aactcagaca ctggcctcaa taggaccaca gtcacgacga tcacagtcta tgcagagcca 1081 cccaaaccct tcatcaccag caacaactcc aaccccgtgg aggatgagga tgctgtagcc 1141 ttaacctgtg aacctgagat tcagaacaca acctacctgt ggtgggtaaa taatcagagc 1201 ctcccggtca gtcccaggct gcagctgtcc aatgacaaca ggaccctcac tctactcagt 1261 gtcacaagga atgatgtagg accctatgag tgtggaatcc agaacgaatt aagtgttgac 1321 cacagcgacc cagtcatcct gaatgtcctc tatggcccag acgaccccac catttccccc 1381 tcatacacct attaccgtcc aggggtgaac ctcagcctct cctgccatgc agcctctaac 1441 ccacctgcac agtattcttg gctgattgat gggaacatcc agcaacacac acaagagctc 1501 tttatctcca acatcactga gaagaacagc ggactctata cctgccaggc caataactca 1561 gccagtggcc acagcaggac tacagtcaag acaatcacag tctctgcgga gctgcccaag 1621 ccctccatct ccagcaacaa ctccaaaccc gtggaggaca aggatgctgt ggccttcacc 1681 tgtgaacctg aggctcagaa cacaacctac ctgtggtggg taaatggtca gagcctccca 1741 gtcagtccca ggctgcagct gtccaatggc aacaggaccc tcactctatt caatgtcaca 1801 agaaatgacg caagagccta tgtatgtgga atccagaact cagtgagtgc aaaccgcagt 1861 gacccagtca ccctggatgt cctctatggg ccggacaccc ccatcatttc ccccccagac 1921 tcgtcttacc tttcgggagc gaacctcaac ctctcctgcc actcggcctc taacccatcc 1981 ccgcagtatt cttggcgtat caatgggata ccgcagcaac acacacaagt tctctttatc 2041 gccaaaatca cgccaaataa taacgggacc tatgcctgtt ttgtctctaa cttggctact 2101 ggccgcaata attccatagt caagagcatc acagtctctg catctggaac ttctcctggt 2161 ctctcagctg gggccactgt cggcatcatg attggagtgc tggttggggt tgctctgata 2221 tagcagccct ggtgtagttt cttcatttca ggaagactga cagttgtttt gcttcttcct 2281 taaagcattt gcaacagcta cagtctaaaa ttgcttcttt accaaggata tttacagaaa 2341 agactctgac cagagatcga gaccatccta gccaacatcg tgaaacccca tctctactaa 2401 aaatacaaaa atgagctggg cttggtggcg cgcacctgta gtcccagtta ctcgggaggc 2461 tgaggcagga gaatcgcttg aacccgggag gtggagattg cagtgagccc agatcgcacc 2521 actgcactcc agtctggcaa cagagcaaga ctccatctca aaaagaaaag aaaagaagac 2581 tctgacctgt actcttgaat acaagtttct gataccactg cactgtctga gaatttccaa 2641 aactttaatg aactaactga cagcttcatg aaactgtcca ccaagatcaa gcagagaaaa 2701 taattaattt catgggacta aatgaactaa tgaggattgc tgattcttta aatgtcttgt 2761 ttcccagatt tcaggaaact ttttttcttt taagctatcc actcttacag caatttgata 2821 aaatatactt ttgtgaacaa aaattgagac atttacattt tctccctatg tggtcgctcc 2881 agacttggga aactattcat gaatatttat attgtatggt aatatagtta ttgcacaagt 2941 tcaataaaaa tctgctcttt gtataacaga aaaa // Her2/Neu Human tyrosine kinase-type receptor (HER2) mRNA, complete cds. ACCESSION M11730 VERSION M11730.1 GI:183986 SEQ ID NO 594 /translation = “MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLD MLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIV RGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQ LCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRT VCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNT DTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKC SKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPL QPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEG LACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPE CQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQ PCPINCTHSCVDLDDKGCPAEQRASPLTSIVSAVVGILLVVVLGVVFGILIKRRQQKI RKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWI PDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVT QLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSP NHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWE LMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFREL VSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGF FCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDG DLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVR PQPPSPREGPLPAARPAGATLERAKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAA PQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV” SEQ ID NO 595 ORIGIN Chromosome 17q21-q22. 1 aattctcgag ctcgtcgacc ggtcgacgag ctcgagggtc gacgagctcg agggcgcgcg 61 cccggccccc acccctcgca gcaccccgcg ccccgcgccc tcccagccgg gtccagccgg 121 agccatgggg ccggagccgc agtgagcacc atggagctgg cggccttgtg ccgctggggg 181 ctcctcctcg ccctcttgcc ccccggagcc gcgagcaccc aagtgtgcac cggcacagac 241 atgaagctgc ggctccctgc cagtcccgag acccacctgg acatgctccg ccacctctac 301 cagggctgcc aggtggtgca gggaaacctg gaactcacct acctgcccac caatgccagc 361 ctgtccttcc tgcaggatat ccaggaggtg cagggctacg tgctcatcgc tcacaaccaa 421 gtgaggcagg tcccactgca gaggctgcgg attgtgcgag gcacccagct ctttgaggac 481 aactatgccc tggccgtgct agacaatgga gacccgctga acaataccac ccctgtcaca 541 ggggcctccc caggaggcct gcgggagctg cagcttcgaa gcctcacaga gatcttgaaa 601 ggaggggtat tgatccagcg gaacccccag ctctgctacc aggacacgat tttgtggaag 661 gacatcttcc acaagaacaa ccagctggct ctcacactga tagacaccaa ccgctctcgg 721 gcctgccacc cctgttctcc gatgtgtaag ggctcccgct gctggggaga gagttctgag 781 gattgtcaga goctgacgcg cactgtctgt gccggtggct gtgcccgctg caaggggcca 841 ctgcccactg actgctgcca tgagcagtgt gctgccggct gcacgggccc caagcactct 901 gactgcctgg cctgcctcca cttcaaccac agtggcatct gtgagctgca ctgcccagcc 961 ctggtcacct acaacacaga cacgtttgag tocatgccca atcccgaggg ccggtataca 1021 ttcggcgcca gctgtgtgac tgcctgtccc tacaactacc tttctacgga cgtgggatcc 1081 tgcaccctcg tctgccccct gcacaaccaa gaggtgacag cagaggatgg aacacagcgg 1141 tgtgagaagt gcagcaagcc ctgtgcccga gtgtgctatg gtctgggcat ggagcacttg 1201 cgagaggtga gggcagttac cagtgccaat atccaggagt ttgctggctg caagaagatc 1261 tttgggagcc tggcatttct gccggagagc tttgatgggg acccagcctc caacactgcc 1321 ccgctccagc cagagcagct ccaagtgttt gagactctgg aagagatcac aggttaccta 1381 tacatctcag catggccgga cagcctgcct gacctcagcg tcttccagaa cctgcaagta 1441 atccggggac gaattctgca caatggcgcc tactcgctga ccctgcaagg gctgggcatc 1501 agctggctgg ggctgcgctc actgagggaa ctgggcagtg gactggccct catccaccat 1561 aacacccacc tctgcttcqt gcacacggtg ccctgggacc agctctttcg gaacccgcac 1621 caagctctgc tccacactgc caaccggcca gaggacgagt gtgtgggcga gggcctggcc 1681 tgccaccagc tgtgcgcccg agggcactgc tggggtccag ggcccaccca gtgtgtcaac 1741 tgcagccagt tccttcgggg ccaggagtgc gtggaggaat gccgagtact gcaggggctc 1801 cccagggagt atgtgaatgc caggcactgt ttgccgtgcc accctgagtg tcagccccag 1861 aatggctcag tgacctgttt tggaccggag gctgaccagt gtgtggcctg tgcccactat 1921 aaqgaccctc ccttctgcgt ggcccgctgc cccagcggtg tgaaacctga cctctcctac 1981 atgcccatct ggaagtttcc agatgaggag ggcgcatgcc agccttgccc catcaactgc 2041 acccactcct gtgtggacct ggatgacaag ggctgccccg cegagoagag agccagccct 2101 ctgacgtcca tcgtctctgc ggtggttggc attctgctgg tcgtggtctt gggggtggtc 2161 tttgggatcc tcatcaagcg acggcagcag aagatccgga agtacacgat gcggagactg 2221 ctgcaggaaa cggagctggt ggagccgctg acacctagcg gagcgatgcc caaccaggcg 2281 cagatgcgga tcctgaaaga gacggagctg aggaaggtga aggtgcttgg atctggcgct 2341 tttggcacag tctacaaggg catctggatc cctgatgggg agaatgtgaa aattccagtg 2401 gccatcaaag tgttgaggga aaacacatcc cccaaagcca acaaagaaat cttagacgaa 2461 gcatacgtga tggctggtgt gggctcccca tatgtctccc gccttctggg catctgcctg 2521 acatccacgg tgcagctggt gacacagctt atgcoctatg gctgcctctt agaccatgtc 2581 cgggaaaacc gcggacgcct gggctcccag gacctgctga actggtgtat gcagattgcc 2641 aaggggatga gctacctgga ggatgtgcgg ctcgtacaca gggacttggc cgctcggaac 2701 gtgctggtca agagtcccaa ccatgtcaaa attacagact tcgggctggc tcggctgctg 2761 gacattgacg agacagagta ccatgcagat gggggcaagg tgcccatcaa gtggatggcg 2821 ctggagtcca ttctccgccg gcggttcacc caccagagtg atgtgtggag ttatggtgtg 2881 actgtgtggg agctgatgac ttttggggcc aaaccttacg atgggatccc agcccgggag 2941 atccctgacc tgctggaaaa gggggagcgg ctgccccagc cccccatctg caccattgat 3001 gtctacatga tcatggtcaa atgttggatg attgactctg aatgtcggcc aagattccgg 3061 gagttggtgt ctgaattctc ccgcatggcc agggaccccc agcgctttgt ggtcatccag 3121 aatgaggact tgggcccagc cagtcccttg gacagcacct tctaccgctc actgctggag 3181 gacgatgaca tgggggacct ggtggatgct gaggagtatc tggtacccca gcagggcttc 3241 ttctgtccag accctgcccc gggcgctggg ggcatggtcc accacaggca ccgcagctca 3301 tctaccagga gtggcggtgg ggacctgaca ctagggctgg agccctctga agaggaggcc 3361 cccaggtctc cactggcacc ctccgaaggg gctggctccg atgtatttga tggtgacctg 3421 ggaatggggg cagccaaggg gctgcaaagc ctccccacac atgaccccag ccctctacag 3481 cggtacagtg aggaccccac agtacccctg ccctctgaga ctgatggcta cgttgccccc 3541 ctgacctgca gcccccagcc tgaatatgtg aaccagccag atgttcggcc ccagccccct 3601 tcgccccgag agggccctct gcctgctgcc cgacctgctg gtgccactct ggaaagggcc 3661 aagactctct ccccagggaa gaatggggtc gtcaaagacg tttttgcctt tgggggtgcc 3721 gtggagaacc ccgagtactt gacaccccag ggaggagctg cccctcagcc ccaccctcct 3781 cctgccttca gcccagcctt cgacaacctc tattactggg accaggaccc accagagcgg 3841 ggggctccac ccagcacctt caaagggaca cctacggcag agaacccaga gtacctgggt 3901 ctggacgtgc cagtgtgaac cagaaggcca agtccgcaga agccctgatg tgtcctcagg 3961 gagcagggaa ggcctgactt ctgctggcat caagaggtgg gagggccctc cgaccacttc 4021 caggggaacc tgccatgcca ggaacctgtc ctaaggaacc ttccttcctg cttgagttcc 4081 cagatggctg gaaggggtcc agcctcgttg gaagaggaac agcactgggg agtctttgtg 4141 gattctgagg ccctgcccaa tgagactcta gggtccagtg gatgccacag cccagcttgg 4201 ccctttcctt ccagatcctg ggtactgaaa gccttaggga agctggcctg agaggggaag 4261 cggccctaag ggagtgtcta agaacaaaag cgacccattc agagactgtc cctgaaacct 4321 agtactgccc cccatgagga aggaacagca atggtgtcag tatccaggct ttgtacagag 4381 tgcttttctg tttagttttt actttttttg ttttgttttt ttaaagacga aataaagacc 4441 caggggagaa tgggtgttgt atggggaggc aagtgtgggg ggtccttctc cacacccact 4501 ttgtccattt gcaaatatat tttggaaaac // H.sapiens mRNA for SCP1 protein. ACCESSION X95654 VERSION X95654.1 GI:1212982 SEQ ID NO 596 /trans1ation = “MEKQKPFALFVPPRSSSSQVSAVKPQTLGGDSTFFKSFNKCTED DLEFPFAKTNLSKNGENIDSDPALQKVNFLPVLEQVGNSDCHYQEGLKDSDLENSEGL SRVFSKLYKEAEKIKKWKVSTEAELRQKESKLQENRKIIEAQRKAIQELQFGNEKVSL KLEEGIQENKDLIKENNATRHLCNLLKETCARSAEKTKKYEYEREETRQVYMDLNNNI EKMITAHGELRVQAENSRLEMHFKLKEDYEKIQHLEQEYKKEINDKEKQVSLLLIQIT EKENKMKDLTFLLEESRDKVNQLEEKTKLQSENLKQSIEKQHHLTKELEDIKVSLQRS VSTQKALEEDLQIATKTICQLTEEKETQMEESNKARAAHSFVVTEFETTVCSLEELLR TEQQRLEKNEDQLKILTMELQKKSSELEEMTKLTNNKEVELEELKKVLGEKETLLYEN KQFEKIAEELKGTEQELIGLLQAREKEVHDLEIQLTAITTSEQYYSKEVKDLKTELEN EKLKNTELTSHCNKLSLENKELTQETSDMTLELKNQQEDINNNKKQEERMLKQIENLQ ETETQLRNELEYVREELKQKRDEVKCKLDKSEENCNNLRKQVENKNKYIEELQQENKA LKKKGTAESKQLNVYEIKVNKLELELESAKQKFGEITDTYQKEIEDKKISEENLLEEV EKAKVIADEAVKLQKEIDKRCQHKIAEMVALMEKHKHQYDKIIEERDSELGLYKSKEQ EQSSLRASLEIELSNLKAELLSVKKQLEIEREEKEKLKREAKENTATLKEKKDKKTQT FLLETPEIYWKLDSKAVPSQTVSRNFTSVDHGISKDKRDYLWTSAKNTLSTPLPKAYT VKTPTKPKLQQRENLNIPIEESKKKRKMAFEFDINSDSSETTDLLSMVSEEETLKTLY RNNNPPASHLCVKTPKKAPSSLTTPGPTLKFGAIRKMREDRWAVIAKMDRKKKLKEAE KLFV” SEQ ID NO 597 ORIGIN 1 gccctcatag accgtttgtt gtagttcgcg tgggaacagc aacccacggt ttcccgatag 61 ttcttcaaag atatttacaa ccgtaacaga gaaaatggaa aagcaaaagc cctttgcatt 121 gttcgtacca ccgagatcaa gcagcagtca ggtgtctgcg gtgaaacctc agaccctggg 181 aggcgattcc actttcttca agagtttcaa caaatgtact gaagatgatt tggagtttcc 241 atttgcaaag actaatctct ccaaaaatgg ggaaaacatt gattcagatc ctgctttaca 301 aaaagttaat ttcttgcccg tgcttgagca ggttggtaat tctgactgtc actatcagga 361 aggactaaaa gactctgatt tggagaattc agagggattg agcagagtgt tttcaaaact 421 gtataaggag gctgaaaaga taaaaaaatg gaaagtaagt acagaagctg aactgagaca 481 gaaagaaagt aagttgcaag aaaacagaaa gataattgaa gcacagcgaa aagccattca 541 ggaactgcaa tttggaaatg aaaaagtaag tttgaaatta gaagaaggaa tacaagaaaa 601 taaagattta ataaaagaga ataatgccac aaggcattta tgtaatctac tcaaagaaac 661 ctgtgctaga tctgcagaaa agacaaagaa atatgaatat gaacgggaag aaaccaggca 721 agtttatatg gatctaaata ataacattga gaaaatgata acagctcatg gggaacttcg 781 tgtgcaagct gagaattcca gactggaaat gcattttaag ttaaaggaag attatgaaaa 841 aatccaacac cttgaacaag aatacaagaa ggaaataaat gacaaggaaa agcaggtatc 901 actactattg atccaaatca ctgagaaaga aaataaaatg aaagatttaa catttctgct 961 agaggaatcc agagataaag ttaatcaatt agaggaaaag acaaaattac agagtgaaaa 1021 cttaaaacaa tcaattgaga aacagcatca tttgactaaa gaactagaag atattaaagt 1081 gtcattacaa agaagtgtga gtactcaaaa ggctttagag gaagatttac agatagcaac 1141 aaaaacaatt tgtcagctaa ctgaagaaaa agaaactcaa atggaagaat ctaataaagc 1201 tagagctgct cattcgtttg tggttactga atttgaaact actgtctgca gcttggaaga 1261 attattgaga acagaacagc aaagattgga aaaaaatgaa gatcaattga aaatacttac 1321 catggagctt caaaagaaat caagtgagct ggaagagatg actaagctta caaataacaa 1381 agaagtagaa cttgaagaat tgaaaaaagt cttgggagaa aaggaaacac ttttatatga 1441 aaataaacaa tttgagaaga ttgctgaaga attaaaagga acagaacaag aactaattgg 1501 tcttctccaa gacagagaga aagaagtaca tgatttggaa atacagttaa ctgccattac 1561 cacaagtgaa cagtattatt caaaagaggt taaagatcta aaaactgagc ttgaaaacga 1621 gaagcttaag aatactgaat taacttcaca ctgcaacaag ctttcactag aaaacaaaga 1681 gctcacacag gaaacaagtg atatgaccct agaactcaag aatcagcaag aagatattaa 1741 taataacaaa aagcaagaag aaaggatgtt gaaacaaata gaaaatcttc aagaaacaga 1801 aacccaatta agaaatgaac tagaatatgt gagagaagag ctaaaacaga aaagagatga 1861 agttaaatgt aaattggaca agagtgaaga aaattgtaac aatttaagga aacaagttga 1921 aaataaaaac aagtatattg aagaacttca gcaggagaat aaggccttga aaaaaaaagg 1981 tacagcagaa agcaagcaac tgaatgttta tgagataaag gtcaataaat tagagttaga 2041 actagaaagt gccaaacaga aatttggaga aatcacagac acctatcaga aagaaattga 2101 ggacaaaaag atatcagaag aaaatctttt ggaagaggtt gagaaagcaa aagtaatagc 2161 tgatgaagca gtaaaattac agaaagaaat tgataagcga tgtcaacata aaatagctga 2221 aatggtagca cttatggaaa aacataagca ccaatatgat aagatcattg aagaaagaga 2281 ctcagaatta ggactttata agagcaaaga acaagaacag tcatcactga gagcatcttt 2341 ggagattgaa ctatccaatc tcaaagctga acttttgtct gttaagaagc aacttgaaat 2401 agaaagagaa gagaaggaaa aactcaaaag agaggcaaaa gaaaacacag ctactcttaa 2461 agaaaaaaaa gacaagaaaa cacaaacatt tttattggaa acacctgaaa tttattggaa 2521 attggattct aaagcagttc cttcacaaac tgtatctcga aatttcacat cagttgatca 2581 tggcatatcc aaagataaaa gagactatct gtggacatct gccaaaaata ctttatctac 2641 accattgcca aaggcatata cagtgaagac accaacaaaa ccaaaactac agcaaagaga 2701 aaacttgaat ataccoattg aagaaagtaa aaaaaagaga aaaatggcct ttgaatttga 2761 tattaattca gatagttcag aaactactga tcttttgagc atggtttcag aagaagagac 2821 attgaaaaca ctgtatagga acaataatcc accagcttct catctttgtg tcaaaacacc 2881 aaaaaaggcc ccttcatctc taacaacccc tggacctaca ctgaagtttg gagctataag 2941 aaaaatgcgg gaggaccgtt gggctgtaat tgctaaaatg gatagaaaaa aaaaactaaa 3001 agaagctgaa aagttatttg tttaatttca gagaatcagt gtagttaagg agcctaataa 3061 cgtgaaactt atagttaata ttttgttctt atttgccaga gccacatttt atctggaagt 3121 tgagacttaa aaaatacttg catgaatgat ttgtgtttct ttatattttt agcctaaatg 3181 ttaactacat attgtctgga aacctgtcat tgtattcaga taattagatg attatatatt 3241 gttgttactt tttcttgtat tcatgaaaac tgtttttact aagttttcaa atttgtaaag 3301 ttagcctttg aatgctagga atgcattatt gagggtcatt ctttattctt tactattaaa 3361 atattttgga tgcaaaaaaa aaaaaaaaaa aaa // Homo sapiens synovial sarcoma, X breakpoint 4 (SSX4), mRNA. ACCESSION NM_005636 VERSION NM_005636.1 GI:5032122 SEQ ID NO 598 /translation = “MNGDDAFARRPRDDAQISEKLRKAFDDIAKYFSKKEWEKMKSSEKIVY VYMKLNYEVMTKLGFKVTLPPFMRSKRAADFHGNDFGNDRNHRNQVERPQMTFG SLQRIFPKIMPKKPAEEENGLKEVPEASGPQNDGKQLCPPGNPSTLEKINKTSGPKRG KHAWTHRLRERKQLVVYEEISDPEEDDE” SEQ ID NO 599 ORIGIN 1 atgaacggag acgacgcctt tgcaaggaga cccagggatg atgctcaaat atcagagaag 61 ttacgaaagg ccttcgatga tattgccaaa tacttctcta agaaagagtg ggaaaagatg 121 aaatcctcgg agaaaatcgt ctatgtgtat atgaagctaa actatgaggt catgactaaa 181 ctaggtttca aggtcaccct cccacctttc atgcgtagta aacgggctgc agacttccac 241 gggaatgatt ttggtaacga tcgaaaccac aggaatcagg ttgaacgtcc tcagatgact 301 ttcggcagcc tccagagaat cttcccgaag atcatgccca agaagccagc agaggaagaa 361 aatggtttga aggaagtgcc agaggcatct ggcccacaaa atgatgggaa acagctgtgc 421 cccccgggaa atccaagtac cttggagaag attaacaaga catctggacc caaaaggggg 481 aaacatgcct ggacccacag actgcgtgag agaaagcagc tggtggttta tgaagagatc 541 agcgaccctg aggaagatga cgagtaactc ccctcg
[0417] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. The entire contents of all patents and publications discussed herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
[0418] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions indicates the exclusion of equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
Claims
1. An isolated epitope, comprising a component selected from the group consisting of:
- (i) a polypeptide having the sequence as disclosed in TABLE 1;
- (ii) an epitope cluster comprising the polypeptide of (i);
- (iii) a polypeptide having substantial similarity to (i) or (ii);
- (iv) a polypeptide having functional similarity to any of (i) through (iii); and
- (v) a nucleic acid encoding the polypeptide of any of (i) through (iv).
2. The epitope of claim 1, wherein the epitope is immunologically active.
3. The epitope of claim 1, wherein the polypeptide is less than about 30 amino acids in length.
4. The epitope of claim 1, wherein the polypeptide is 8 to 10 amino acids in length.
5. The epitope of claim 1, wherein the substantial or functional similarity comprises addition of at least one amino acid.
6. The epitope of claim 5, wherein the at least one additional amino acid is at an N-terminus of the polypeptide.
7. The epitope of claim 1, wherein the substantial or functional similarity comprises a substitution of at least one amino acid.
8. The epitope of claim 1, the polypeptide having affinity to an HLA-A2 molecule.
9. The epitope of claim 8, wherein the affinity is determined by an assay of binding.
10. The epitope of claim 8, wherein the affinity is determined by an assay of restriction of epitope recognition.
11. The epitope of claim 8, wherein the affinity is determined by a prediction algorithm.
12. The epitope of claim 1, the polypeptide having affinity to an HLA-B7 or HLA-B51 molecule.
13. The epitope of claim 1, wherein the polypeptide is a housekeeping epitope.
14. The epitope of claim 1, wherein the polypeptide corresponds to an epitope displayed on a tumor cell.
15. The epitope of claim 1, wherein the polypeptide corresponds to an epitope displayed on a neovasculature cell.
16. The epitope of claim 1, wherein the polypeptide is an immune epitope.
17. The epitope of claim 1 wherein the epitope is a nucleic acid.
18. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.
19. The composition of claim 18, where the adjuvant is a polynucleotide.
20. The composition of claim 19 wherein the polynucleotide comprises a dinucleotide.
21. The composition of claim 20 wherein the dinucleotide is CpG.
22. The composition of claim 18, wherein the adjuvant is encoded by a polynucleotide.
23. The composition of claim 18 wherein the adjuvant is a cytokine.
24. The composition of claim 23 wherein the cytokine is GM-CSF.
25. The composition of claim 18 further comprising a professional antigen-presenting cell (pAPC).
26. The composition of claim 25, wherein the pAPC is a dendritic cell.
27. The composition of claim 18, further comprising a second epitope.
28. The composition of claim 27, wherein the second epitope is a polypeptide.
29. The composition of claim 27, wherein the second epitope is a nucleic acid.
30. The composition of claim 27, wherein the second epitope is a housekeeping epitope.
31. The composition of claim 27, wherein the second epitope is an immune epitope.
32. A pharmaceutical composition comprising the nucleic acid of claim 1 and a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.
33. A recombinant construct comprising the nucleic acid of claim 1.
34. The construct of claim 33, further comprising a plasmid, a viral vector, or an artificial chromosome.
35. The construct of claim 33, further comprising a sequence encoding at least one feature selected from the group consisting of a second epitope, an IRES, an ISS, an NIS, and ubiquitin.
36. A purified antibody that specifically binds to the epitope of claim 1.
37. A purified antibody that specifically binds to a peptide-MHC protein complex comprising the epitope of claim 1.
38. The antibody of claim 36 or claim 37, wherein the antibody is a monoclonal antibody.
39. A multimeric MHC-peptide complex comprising the epitope of claim 1.
40. An isolated T cell expressing a T cell receptor specific for an MHC-peptide complex, the complex comprising the epitope of claim 1.
41. The T cell of claim 40, produced by an in vitro immunization.
42. The T cell of claim 40, isolated from an immunized animal.
43. A T cell clone comprising the T cell of claim 40.
44. A polyclonal population of T cells comprising the T cell of claim 40.
45. A pharmaceutical composition comprising the T cell of claim 40 and a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.
46. An isolated protein molecule comprising the binding domain of a T cell receptor specific for an MHC-peptide complex, the complex comprising the epitope of claim 1.
47. The protein of claim 46, wherein the protein is multivalent.
48. An isolated nucleic acid encoding the protein of claim 46.
49. A recombinant construct comprising the nucleic acid of claim 48.
50. A host cell expressing the recombinant construct, the construct comprising the nucleic acid of claim 1, or the construct encoding a protein molecule comprising the binding domain of a T cell receptor specific for an MHC-peptide complex.
51. The host cell of claim 50, wherein the host cell is a dendritic cell, macrophage, tumor cell, or tumor-derived cell.
52. The host cell of claim 50, wherein the host cell is a bacterium, fungus, or protozoan.
53. A pharmaceutical composition comprising the host cell of claim 50 and a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.
54. A vaccine or immunotherapeutic composition comprising at least one component selected from the group consisting of the epitope of claim 1; the composition of claim 18, 32, or 45, the construct of claim 33; the T cell of claim 40, a host cell expressing a recombinant construct comprising a nucleic acid encoding a T cell receptor binding domain specific for an MHC-peptide complex and a composition comprising the same, and a host cell expressing a recombinant construct comprising the nucleic acid of claim 1 and a composition comprising the same.
55. A method of treating an animal, comprising:
- administering to an animal the vaccine or immunotherapeutic composition of claim 54.
56. The method of claim 55, wherein the administering step comprises a mode of delivery selected from the group consisting of transdermal, intranodal, perinodal, oral, intravenous, intradermal, intramuscular, intraperitoneal, mucosal, aerosol inhalation, and instillation.
57. The method of claim 55, further comprising a step of assaying to determine a characteristic indicative of a state of a target cell or target cells.
58. The method of claim 57, comprising a first assaying step and a second assaying step, wherein the first assaying step precedes the administering step, and wherein the second assaying step follows the administering step.
59. The method of claim 58, further comprising a step of comparing the characteristic determined in the first assaying step with the characteristic determined in the second assaying step to obtain a result.
60. The method of claim 59, wherein the result is selected from the group consisting of: evidence of an immune response, a diminution in number of target cells, a loss of mass or size of a tumor comprising target cells, a decrease in number or concentration of an intracellular parasite infecting target cells.
61. A method of evaluating immunogenicity of a vaccine or immunotherapeutic composition, comprising:
- administering to an animal the vaccine or immunotherapeutic composition of claim 54; and
- evaluating immunogenicity based on a characteristic of the animal.
62. The method of claim 61, wherein the animal is HLA-transgenic.
63. A method of evaluating immunogenicity, comprising:
- in vitro stimulation of a T cell with the vaccine or immunotherapeutic composition of claim 54; and
- evaluating immunogenicity based on a characteristic of the T cell.
64. The method of claim 63, wherein the stimulation is a primary stimulation.
65. A method of making a passive/adoptive immunotherapeutic, comprising:
- combining the T cell of claim 40, or a host cell expressing a recombinant construct comprising a nucleic acid encoding a T cell receptor binding domain specific for an MHC-peptide complex, or a host cell expressing a recombinant construct comprising the nucleic acid of claim 1 with a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.
66. A method of determining specific T cell frequency comprising the step of contacting T cells with a MHC-peptide complex comprising the epitope of claim 1.
67. The method of claim 66, wherein the contacting step comprises at least one feature selected from the group consisting of immunization, restimulation, detection, and enumeration.
68. The method of claim 66, further comprising ELISPOT analysis, limiting dilution analysis, flow cytometry, in situ hybridization, the polymerase chain reaction or any combination thereof.
69. A method of evaluating immunologic response, comprising the method of claim 66 carried out prior to and subsequent to an immunization step.
70. A method of evaluating immunologic response, comprising:
- determining frequency, cytokine production, or cytolytic activity of T cells, prior to and subsequent to a step of stimulation with MHC-peptide complexes comprising the epitope of claim 1.
71. A method of diagnosing a disease comprising:
- contacting a subject tissue with at least one component selected from the group consisting of the T cell of claim 40, the host cell of claim 50, the antibody of claim 36, and the protein of claim 46; and
- diagnosing the disease based on a characteristic of the tissue or of the component.
72. The method of claim 71, wherein the contacting step takes place in vivo.
73. The method of claim 71, wherein the contacting step takes place in vitro.
74. A method of making a vaccine, comprising:
- combining at least one component selected from the group consisting of the epitope of claim 1; the composition of claim 18, 32, 45, or 53; the construct of claim 33; the T cell of claim 40, and the host cell of claim 50, with a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient.
75. A computer readable medium having recorded thereon the sequence of any one of SEQ ID NOS: 1-602, in a machine having a hardware or software that calculates the physical, biochemical, immunologic, or molecular genetic properties of a molecule embodying said sequence.
76. A method of treating an animal comprising combining the method of claim 55 combined with at least one mode of treatment selected from the group of radiation therapy, chemotherapy, biochemotherapy, and surgery.
77. An isolated polypeptide comprising an epitope cluster from a target-associated antigen having the sequence as disclosed in Tables 25-44, wherein the amino acid sequence consists of not more than about 80% of the amino acid sequence of the antigen.
78. A vaccine or immunotherapeutic product comprising the polypeptide of claim 78.
79. An isolated polynucleotide encoding the polypeptide of claim 78.
80. A vaccine or immunotherapeutic product comprising the polynucleotide of claim 80.
81. The polynucleotide of claim 79 or 80, wherein the polynucleotide is DNA.
82. The polynucleotide of claim 79 or 80, wherein the polynucleotide is RNA.
Type: Application
Filed: Apr 4, 2002
Publication Date: Nov 27, 2003
Inventors: John J. L. Simard (Northridge, CA), David C. Diamond (West Hills, CA), Liping Liu (Woodland Hills, CA), Zhidong Xie (Woodland Hills, CA)
Application Number: 10117937
International Classification: C12Q001/68; G01N033/574; A61K048/00; A61K038/17;
